polynucleotides, compositions and methods for genome editing

专利摘要:
Compositions and methods for gene editing. In some embodiments, a polynucleotide encoding Cas9 is provided that may provide one or more of improved editing efficiency, reduced immunogenicity, or other benefits.
公开号:BR112020005323A2
申请号:R112020005323-6
申请日:2018-09-28
公开日:2020-09-24
发明作者:Christian Dombrowski；Jonathan Douglas Finn；Amy Madison Rhoden SMITH；Seth C. Alexander
申请人:Intellia Therapeutics, Inc.；
IPC主号:

专利说明:

[0001] [0001] This application claims priority benefit to United States Interim Application 62/556,144, which was filed on September 29, 2017 and which has been incorporated by reference in its entirety.
[0002] [0002] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on September 28, 2018, is named 2018-09-28_01155-0020-00PCT_ST25.txt and is 963,200 bytes in size.
[0003] [0003] The present disclosure relates to polynucleotides, compositions and methods for genome editing that involve RNA-guided DNA binding agents such as CRISPR-Cas systems and subunits thereof.
[0004] [0004] RNA-guided DNA binding agents such as CRISPR-Cas systems can be used for targeted genome editing, including eukaryotic cells and in vivo. This editing has been shown to be able to inactivate certain deleterious alleles or correct certain deleterious point mutations. The agent can be expressed in situ by providing mRNA that encodes it. Existing approaches may, however, provide less editing efficiency than desired or may be immunogenic undesirable, for example, may cause an undesirable elevation in cytokine levels.
[0005] [0005] Thus, there is a need for improved polynucleotides, compositions and methods for genome editing. The present disclosure is intended to provide compositions and methods for genome editing that provide one or more benefits, such as at least one of improved editing efficiency or reduced immunogenicity (e.g., reduced elevation of cytokines after administration) or at least to provide the public with a useful choice. In some embodiments, a polynucleotide encoding an RNA-guided DNA-binding agent is provided, wherein one or more of its codon usage, non-coding sequence (e.g., a UTR), heterologous domain (e.g., NLS ) and/or nucleotide content differs from existing polynucleotides in a manner disclosed herein. These features have been found to provide benefits such as those described above. In some embodiments, the improved editing efficiency occurs or is specific to a mammalian organ or cell type, such as the liver or hepatocytes. SUMMARY
[0006] [0006] Embodiment 1 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content.
[0007] [0007] Embodiment 2 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has a uridine dinucleotide content that varies from its minimum uridine dinucleotide content. uridine at 150% of the minimum uridine content.
[0008] [0008] Embodiment 3 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has an adenine content ranging from its minimum uridine content to 150% of the minimum adenine content.
[0009] [0009] Embodiment 4 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has an adenine dinucleotide content that varies from its minimum adenine dinucleotide content. adenine at 150% of the minimum adenine content.
[0010] [0010] Embodiment 5 is an mRNA comprising a sequence having at least 90% identity to any of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20 , 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175, wherein the mRNA comprises an open reading frame encoding a DNA-binding agent guided by RNA.
[0011] [0011] Embodiment 6 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has at least 90% identity to any of SEQ ID NO: 1 , 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175 for at least its first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides.
[0012] [0012] Modality 7 is the mRNA of any of the above embodiments comprising the open reading frame consisting of a set of codons of which at least 75% of the codons are (i) codons listed in Table 1, Table 2 or Table 3 , or (ii) a set of codons listed in Table 4.
[0013] [0013] Modality 8 is an mRNA encoding an RNA-guided DNA binding agent, characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, in which the reading frame open consists of a set of codons of which at least 75% of the codons are codons listed in Table 1, Table 2, Table 3 or (ii) a set of codons listed in Table 4.
[0014] [0014] Modality 9 is modality 7 or 8 mRNA, where the open reading frame consists of a set of codons of which at least 75% of the codons are codons from the low U 1 set defined in Table 4.
[0015] [0015] Modality 10 is modality 7 or 8 mRNA, where the open reading frame consists of a set of codons of which at least 75% of the codons are codons from set A below defined in Table 4.
[0016] [0016] Modality 11 is modality 7 or 8 mRNA, where the open reading frame consists of a set of codons of which at least 75% of the codons are codons from the low A/U set defined in Table 4.
[0017] [0017] Modality 9 is modality 7 or 8 mRNA, where the open reading frame consists of a set of codons of which at least 75% of the codons are codons from the long half-life set defined in Table 4.
[0018] [0018] Modality 13 is the mRNA of any of modalities 7 to 12, wherein at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are (i) listed codons in Table 1, Table 2 or Table 3 or (ii) a set of codons listed in Table 4.
[0019] [0019] Embodiment 14 is the mRNA of any one of Embodiments 1 to 5 or 7 to 13, wherein the open reading frame has at least 90% identity to any of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175 during at least their first few 30, 50, 70, 100, 150, 200, 250 or 300 nucleotides.
[0020] [0020] Embodiment 15 is the mRNA of any of the foregoing embodiments, wherein the open reading frame has at least 90% identity to any of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175 for at least the first 10%, 12%, 15%, 20%, 25% , 30% or 35% of your streak.
[0021] [0021] Embodiment 16 is the mRNA of any one of Embodiments 1 to 4 or 6 to 15, wherein the mRNA comprises a sequence with at least 90% identity to any of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27,
[0022] [0022] Embodiment 17 is the mRNA of any of the foregoing embodiments, wherein the open reading frame has a uridine dinucleotide content that ranges from its minimum uridine dinucleotide content to 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum uridine dinucleotide content.
[0023] [0023] Embodiment 18 is the mRNA of any of the foregoing embodiments, wherein the open reading frame has a uridine content that ranges from its minimum uridine content to 101%, 102%, 103%, 105%, 110 %, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum uridine content.
[0024] [0024] Embodiment 19 is the mRNA of any of the foregoing embodiments, wherein the open reading frame has an adenine content ranging from its minimum uridine content to 101%, 102%, 103%, 105%, 110 %, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum adenine content.
[0025] [0025] Embodiment 20 is the mRNA of any of the foregoing embodiments, wherein the open reading frame has an adenine dinucleotide content that ranges from its minimum adenine dinucleotide content to 101%, 102%, 103%, 105 %, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum content of adenine dinucleotide.
[0026] [0026] Embodiment 21 is the mRNA of any of the foregoing embodiments, wherein it comprises a 5' UTR with at least 90% identity to any of SEQ ID NOs: 32, 34, 36, 38, 41, or 75- 77.
[0027] [0027] Embodiment 22 is the mRNA of any of the foregoing embodiments, wherein a 3' UTR has at least 90% identity to any of SEQ ID NOs: 33, 35, 37, 39, or 40.
[0028] [0028] Modality 23 is modality 21 or 22 mRNA, wherein the mRNA comprises a 5' UTR and a 3' UTR from the same source.
[0029] [0029] Modality 24 is the mRNA of any of the previous modalities, in which it comprises a 5' cap selected from Cap0, Cap1 and Cap2.
[0030] [0030] Embodiment 25 is the mRNA of any of the foregoing embodiments, wherein the open reading frame has codons that enhance translation of the mRNA in a mammal.
[0031] [0031] Modality 26 is modality 25 mRNA, wherein the open reading frame has codons that enhance translation of the mRNA in a specific mammalian organ.
[0032] [0032] Modality 27 is modality 26 mRNA, where the organ is liver.
[0033] [0033] Embodiment 28 is the mRNA of any one of Embodiments 25 to 27, wherein the mammal is a human.
[0034] [0034] Embodiment 29 is the mRNA of any one of embodiments 25 to 28, wherein the codons enhance translation of the mRNA in the mammal relative to translation of an mRNA comprising an ORF with a sequence consisting of SEQ ID NO: 5 .
[0035] [0035] Embodiment 30 is the mRNA of any of the foregoing embodiments, wherein when the mRNA is administered to a mammal in a pharmaceutical composition, the mammal exhibits a cytokine response at least 5-fold lower than a mammal that has been administered with an mRNA comprising an ORF encoding a Cas9 nuclease with greater than 150% of the minimum uridine content.
[0036] [0036] Embodiment 31 is modality 30 mRNA, wherein the mRNA comprising the ORF encoding the Cas9 nuclease with more than 150% of the minimum uridine content has a sequence consisting of SEQ ID NO: 5.
[0037] [0037] Modality 132 is mRNA from any of the foregoing modalities, wherein the RNA-guided DNA binding agent has double-stranded endonuclease activity.
[0038] [0038] Modality 33 is modality 32 mRNA, wherein the RNA-guided DNA binding agent comprises a Cas clevase.
[0039] [0039] Modality 34 is the mRNA of any of the foregoing embodiments, in which the RNA-guided DNA binding agent has nickase activity.
[0040] [0040] Modality 35 is modality 34 mRNA, wherein the RNA-guided DNA binding agent comprises a Cas nickase.
[0041] [0041] Embodiment 36 is the mRNA of any one of modalities 1 to 31, wherein the RNA-guided DNA binding agent comprises a dCas DNA binding domain.
[0042] [0042] Embodiment 37 is the mRNA of any one of embodiments 33 or 35 to 36, wherein the Cas clevase, Cas nickase, or dCas DNA binding domain is a Cas9 clevase, Cas9 nickase, or dCas9 DNA binding domain .
[0043] [0043] Embodiment 38 is the mRNA of any of the foregoing embodiments, wherein the encoded RNA-guided DNA binding agent comprises a nuclear localization signal (NLS).
[0044] [0044] Modality 39 is modality 38 mRNA, in which the NLS is linked to the C-terminus of the RNA-guided DNA binding agent.
[0045] [0045] Modality 40 is modality 38 mRNA, in which the NLS is linked to the N-terminus of the RNA-guided DNA binding agent.
[0046] [0046] Embodiment 41 is the mRNA of any one of modalities 38 to 40, wherein the NLS comprises a sequence with at least 80%, 85%, 90%, or 95% identity to any of SEQ ID NOs: 78 -91.
[0047] [0047] Embodiment 42 is the mRNA of any one of embodiments 38 to 40, wherein the NLS comprises the sequence of any one of SEQ ID NOs: 78-91.
[0048] [0048] Modality 43 is the mRNA of any one of modalities 38 to 42, wherein the NLS is encoded by a sequence with at least 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence of any one of SEQ ID NOs: 92-104.
[0049] [0049] Embodiment 44 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 90% identity to SEQ ID NO: 4, 7, or 9.
[0050] [0050] Embodiment 45 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 95% identity to SEQ ID NO: 4, 7, or 9.
[0051] [0051] Embodiment 46 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 98% identity to SEQ ID NO: 4, 7, or 9.
[0052] [0052] Embodiment 47 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence 100% identical to SEQ ID NO: 4, 7, or 9.
[0053] [0053] Embodiment 48 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 90% identity to SEQ ID NO: 111, 114, or 117.
[0054] [0054] Embodiment 49 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 95% identity to SEQ ID NO: 111, 114, or 117.
[0055] [0055] Embodiment 50 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 98% identity to SEQ ID NO: 111, 114, or 117.
[0056] [0056] Embodiment 51 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence 100% identical to SEQ ID NO: 112, 122, or 125.
[0057] [0057] Embodiment 52 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 90% identity to SEQ ID NO: 112, 122, or 125.
[0058] [0058] Embodiment 53 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 95% identity to SEQ ID NO: 112, 122, or 125.
[0059] [0059] Embodiment 54 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 90% identity to SEQ ID NOs: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.
[0060] [0060] Embodiment 55 is the mRNA of any one of Embodiments 37 to 43, wherein it comprises a sequence with at least 95% identity to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[0061] [0061] Embodiment 56 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence with at least 98% identity to SEQ ID NOs: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.
[0062] [0062] Embodiment 57 is the mRNA of any one of Embodiments 37 to 43, wherein the mRNA comprises a sequence 100% identical to SEQ ID NOs: 10, 11, 12, 14, 15, 17, 18, 20, 21 , 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.
[0063] [0063] Embodiment 58 is the mRNA of any one of embodiments 37 to 57, wherein the mRNA encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 8 or 186-196.
[0064] [0064] Embodiment 59 is the mRNA of any of the foregoing embodiments, wherein the RNA-guided DNA binding agent further comprises a heterologous functional domain.
[0065] [0065] Modality 60 is modality 59 mRNA, wherein the heterologous functional domain is a Fokl nuclease.
[0066] [0066] Modality 61 is modality 59 mRNA, wherein the heterologous functional domain is a transcriptional regulatory domain.
[0067] [0067] Embodiment 62 is the mRNA of any of the foregoing embodiments, wherein when an effective amount of the mRNA is administered to a mammal together with a guide RNA that targets the mammal's TTR gene in a pharmaceutical composition comprising lipid nanoparticles, a indel is formed at the TTR locus in at least 50% of genomic DNA obtained from mammalian hepatocytes.
[0068] [0068] Embodiment 63 is the mRNA of any of the foregoing embodiments, wherein when an effective amount of the mRNA is administered to a mammal together with a guide RNA that targets the mammal's TTR gene in a pharmaceutical composition comprising lipid nanoparticles, the mammalian serum TTR concentration is reduced by at least 50%.
[0069] [0069] Embodiment 64 is the mRNA of any of the foregoing embodiments, wherein at least 10% of the uridine is replaced by a modified uridine.
[0070] [0070] Modality 65 is modality 64 mRNA, wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.
[0071] [0071] Modality 66 is modality 64 mRNA, wherein the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.
[0072] [0072] Modality 67 is modality 64 mRNA, wherein the modified uridine is N1-methyl-pseudouridine.
[0073] [0073] Modality 68 is modality 64 mRNA, wherein the modified uridine is 5-methoxyuridine.
[0074] [0074] Embodiment 69 is the mRNA of any one of modalities 64 to 68, wherein 15% to 45% of the uridine is replaced by the modified uridine.
[0075] [0075] Modality 70 is the mRNA of any one of modalities 64 to 68, wherein at least 20% or at least 30% of the uridine is replaced by the modified uridine.
[0076] [0076] Modality 71 is modality 70 mRNA, wherein at least 80% or at least 90% of the uridine is replaced by the modified uridine.
[0077] [0077] Modality 72 is modality 70 mRNA, wherein 100% uridine is replaced by the modified uridine.
[0078] [0078] Embodiment 73 is the mRNA of any one of embodiments 64 to 72, wherein when an effective amount of the mRNA is administered to a mammal together with a guide RNA that targets the mammal's TTR gene in a pharmaceutical composition comprising lipid nanoparticles , an indel is formed at the TTR locus in at least 70% or at least 90% of the genomic DNA obtained from mammalian hepatocytes.
[0079] [0079] Embodiment 74 is the mRNA of any one of modalities 64 to 73, wherein when the mRNA is administered to a mammal together with a guide RNA that targets the mammal's TTR gene in a pharmaceutical composition comprising lipid nanoparticles, the concentration mammalian serum TTR is reduced by at least 70% or at least 90%.
[0080] [0080] Modality 75 is modality 62, 63, 71 or 72 mRNA where the animal is a mouse and the guide RNA has a sequence consisting of SEQ ID NO: 42.
[0081] [0081] Modality 76 is the mRNA of modality 62, 63, 71 or 72,
[0082] [0082] Embodiment 77 is the mRNA of any of the foregoing embodiments, wherein the mRNA comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.
[0083] [0083] Embodiment 78 is the mRNA of any of the foregoing embodiments, wherein the mRNA comprises a sequence having at least 95% identity to any of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.
[0084] [0084] Embodiment 79 is the mRNA of any of the foregoing embodiments, wherein the mRNA comprises a sequence having at least 98% identity to any of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.
[0085] [0085] Embodiment 80 is the mRNA of any of the foregoing embodiments, wherein the mRNA comprises a sequence with at least 99% identity to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.
[0086] [0086] Embodiment 81 is the mRNA of any of the foregoing embodiments, wherein the mRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 176- 185.
[0087] [0087] Embodiment 82 is an expression construct comprising a promoter operably linked to a sequence encoding an mRNA as defined in any of the foregoing embodiments.
[0088] [0088] Modality 83 is a plasmid comprising the expression construct of modality 82.
[0089] [0089] Modality 84 is a host cell comprising the modality 82 expression construct or the modality 83 plasmid.
[0090] [0090] Modality 85 is a method for preparing an mRNA comprising contacting the expression construct as defined in modality 82 or the plasmid as defined in modality 83 with an RNA polymerase under conditions permissive for transcription of the mRNA.
[0091] [0091] Modality 86 is the method of modality 85, in which the contact step is performed in vitro.
[0092] [0092] Embodiment 87 is a composition comprising an mRNA as defined in any one of Embodiments 1 to 81 and at least one guide RNA.
[0093] [0093] Embodiment 88 is a lipid nanoparticle comprising an mRNA according to any one of Embodiments 1 to 81.
[0094] [0094] Embodiment 89 is a pharmaceutical composition comprising an mRNA as defined in any one of Embodiments 1 to 81 and a pharmaceutically acceptable carrier.
[0095] [0095] Modality 90 is the lipid nanoparticle of modality 88 or the pharmaceutical composition of modality 89, further comprising at least one guide RNA.
[0096] [0096] Embodiment 91 is the composition or lipid nanoparticle, according to any one of claims 87 to 90, wherein at least one guide RNA directs TTR.
[0097] [0097] Embodiment 92 is a method for editing or modifying a genome of a target gene comprising contacting a cell with the mRNA, expression construct, composition or lipid nanoparticle of any one of claims 1-83 or 87-91 .
[0098] [0098] Embodiment 93 is the use of the mRNA, expression construct, composition or lipid nanoparticle of any one of claims 1 to 83 or 87 to 91 for genome editing or modification of a target gene.
[0099] [0099] Modality 94 is the use of the mRNA, expression construct, composition or lipid nanoparticle of any one of claims 1 to 83 or 87 to 91 for the manufacture of a drug for editing or modifying the genome of a target gene.
[00100] [00100] Embodiment 95 is the method or use of any one of claims 92 to 94, wherein the editing or modification of the target gene genome takes place in a liver cell.
[00101] [00101] Embodiment 96 is the method or use of claim 95, wherein the liver cell is a hepatocyte.
[00102] [00102] Embodiment 97 is the method or use of any one of claims 92 to 96, wherein the editing or modification of the target gene genome is in vivo.
[00103] [00103] Embodiment 98 is the method or use of any one of claims 92 to 97, wherein the editing or modification of the target gene genome is in an isolated or cultured cell.
[00104] [00104] See the Sequence Table below for the sequences themselves. Transcript sequences generally include GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCapTM. Therefore, the first three nucleotides can be modified for use with other capping approaches, such as the Vaccinia uptake enzyme. Promoters and poly-A sequences are not included in transcriptional sequences. A promoter such as a T7 promoter (SEQ ID NO: 31) and a poly-A sequence such as SEQ ID NO: 62 or 63 can be appended to the disclosed transcript sequences at the 5' and 3' ends, respectively. Most nucleotide sequences are provided as DNA, but can be easily converted to RNA by changing Ts to Us. BRIEF DESCRIPTION OF THE FIGURES
[00105] [00105] Figures 1A-1D show levels of IFN alpha, IL-6, TNF alpha and MCP-1 after administration of PBS or lipid nanoparticles (LNP) LNP417-LNP421 at 0.5 or 1 mg/kg (mpk).
[00106] [00106] Figures 2A-2B show serum levels of TTR and percentage of liver editing after administration of the PBS or LNP formulation LNP417-LNP421 at 0.5 or 1 mpk.
[00107] [00107] FIGURE 3 shows in vitro transcription (IVT) yields for transcription from Cas9 DNA constructs. Transcription was performed with unmodified uridine-5'-triphosphate (UTP) or N1-methyl-pseudo-UTP alone (0 on the horizontal axis), mixed with an indicated ratio of 5-methoxy UTP (20-80 on the horizontal axis). ) or with 100% 5-methoxy UTP (100). For each set of three slashes, the left slash used N1-methyl-pseudo-UTP and/or 5-methoxy UTP and SEQ ID NO: 2; center bar used unmodified UTP and/or 5-methoxy UTP and SEQ ID NO: 2; and the right slash used unmodified UTP and/or 5-methoxy UTP and SEQ ID NO: 1.
[00108] [00108] FIGURE 4 shows the mRNA purity of in vitro transcription (IVT) results for Cas9 (SEQ ID NO: 2) and Cas9-optimized DNA constructs (SEQ ID NO: 1). Transcription was performed from the Cas9 sequence of SEQ ID NO: 2 with unmodified uridine-5'-triphosphate (UTP) (squares) or with N1-methyl-pseudo-UTP (dark circles) alone (0) or mixed with the indicated proportion of 5-methoxy UTP (20-80) or with 100% 5-methoxy UTP (100). Transcription was performed from the Cas9 sequence of SEQ ID NO: 1 (light circles) with unmodified UTP (0) or mixed with an indicated ratio of 5-methoxy UTP (20-80) or with 100% 5-methoxy UTP (100 µM) Each coding sequence included a nuclear localization signal.
[00109] [00109] Figures 5A-5D show staining results on anti-dsRNA antibody. Results are with double-stranded RNA control (A), Cas9 transcribed in the presence of UTP and/or 5-methoxy UTP (B), Cas9 mRNA sequence comprising SEQ ID NO: 4 transcribed in the presence of UTP and/or 5-methoxy UTP (C) and Cas9 transcribed in the presence of N1-methyl-pseudo-UTP and/or 5-methoxy UTP (D). Panels (B) - (D) were performed with transcripts containing 0% to 100% 5-methoxy UTP and 100% to 0% UTP or N1-methyl UTP.
[00110] [00110] Figures 6A and 6B show in vitro editing efficiency of mRNAs in Neuro 2A cells treated with Cas9 mRNA, presented as percent edit (A) or EC50 edit (B). The effect of increasing concentrations of 5-methoxy-UTP on Cas9 mRNA was evaluated. Transcription was performed from the Cas9 sequence of SEQ ID NO: 2 with N1-methyl-pseudo-UTP (left set in A; dark circles in B) or with unmodified uridine-5'-triphosphate (UTP) (central set in A; squares in B) alone (0) or mixed with an indicated proportion of 5-methoxy UTP (20-80), or with 100% 5-methoxy UTP (100). Transcription was performed from the Cas9 sequence of SEQ ID NO: 1 (set right in A; light circles in B) with unmodified UTP (0) or mixed with an indicated ratio of 5-methoxy UTP
[00111] [00111] Figures 7A-7D show serum cytokine levels 4 hours post-dose for LNP formulations LNP720-LNP724. The asterisk in FIGURE 7A indicates that at least one individual measurement was below the detection limit.
[00112] [00112] Figures 8A and 8B show serum levels of TTR (A) and percentage of TTR editing in the liver (B) 7 days after dosing with the LNP formulations LNP720-LNP724. The asterisk in FIGURE 8A indicates that at least one individual measurement was below the detection limit.
[00113] [00113] FIGURE 9 shows the percentage of TTR editing in the spleen 7 days after dosing with the LNP LNP720-LNP724 formulations at 1 mpk.
[00114] [00114] FIGURE 10 shows the percentage of TTR editing in mouse primary hepatocytes (PMH) with LNP formulations LNP720-LNP724 and LNP685.
[00115] [00115] Figures 11A and 11B show serum levels of TTR after dosing of formulations comprising Cas9 mRNAs in which the ORFs had sequences of SEQ ID NO: 5 or 4. TTR data are presented as serum levels (A) or percentage in relation to TTR levels in animals treated with TSS (B).
[00116] [00116] FIGURE 12 shows the percentage of TTR editing in the liver after dosing of formulations comprising a Cas9 mRNA in which the ORF had the sequence of SEQ ID NO: 5 or 4 to 5 mpk or 2 mpk.
[00117] [00117] Figures 13A-E show serum levels of TTR and the percentage of TTR editing in the liver following dosing of the indicated LNP formulations.
[00118] [00118] FIGURE 14 shows the percentage of TTR editing in mouse primary hepatocytes (PMH) treated with 0.3, 1, 3 or 10 ng of LNP815-821, 823 or 824.
[00119] [00119] Figures 15A-B show serum levels of TTR after dosing of LNP formulations containing a Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO: 5 or 4 in the ratios and amounts indicated in Guide:Cas9.
[00120] [00120] Figures 16A-B show percentage of TTR editing in the liver following dosing of LNP formulations containing a Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO: 5 or 4 in the ratios and values indicated in Guide :Cas9.
[00121] [00121] Figures 17A-B shows the percentage of TTR editing in the spleen following dosing of LNP formulations containing a Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO: 5 or 4 in the ratios and amounts indicated in Guide:Cas9.
[00122] [00122] FIGURE 18 shows a Western blot of Cas9 expression in the liver after dosing of LNP formulations containing a Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO: 5 or 4 in the indicated ratios of Guide:Cas9 .
[00123] [00123] Figures 19A-B show serum levels of TTR after dosing of the indicated LNP formulations in the indicated amounts.
[00124] [00124] FIGURE 20 shows the percentage of TTR editing in the liver after dosing the indicated LNP formulations in the indicated amounts.
[00125] [00125] Figures 21A-C show liver edit levels (A) and serum TTR (B in µg/ml; C as percentage of TSS control) after dosing the indicated LNP formulations in the indicated amounts.
[00126] [00126] Figures 22A-D show serum TTR and edit results after dosing LNP formulations in the indicated ratios and amounts.
[00127] [00127] FIGURE 23 shows the expression of Cas9 protein in Hep2G cells after treatment with Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO indicated.
[00128] [00128] FIGURE 24 shows the percentage of editing in HepG2 cells after treatment with Cas9 mRNA, where the ORFs had the sequence of SEQ ID NO indicated at the indicated concentrations.
[00129] [00129] FIGURE 25 shows the expression of Cas9 in the liver following dosing of LNP formulations with Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO indicated.
[00130] [00130] FIGURE 26 shows results of in vivo editing at the TTR locus after dosing LNP formulations with Cas9 mRNA in which the ORFs had the indicated sequence of SEQ ID NO.
[00131] [00131] Figures 27A-B show serum TTR (A) and serum TTR (%TSS) (B) after dosing of LNP formulations with Cas9 mRNA in which the ORFs had the indicated sequence of SEQ ID NO.
[00132] [00132] FIGURE 28 shows hepatic editing in vivo after dosing of LNP formulations with Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO indicated in the indicated amounts.
[00133] [00133] Figures 29A-B show serum levels of TTR (A) and serum TTR (%TSS) (B) after dosing of LNP formulations with Cas9 mRNA in which the ORFs had the sequence of SEQ ID NO indicated in the indicated quantities.
[00134] [00134] Figures 30A-B show serum levels of TTR (A) and % edit in liver (B) after dosing of LNP formulations with Cas9 mRNA in which transcripts had the sequence of SEQ ID NO indicated.
[00135] [00135] FIGURE 31 shows the percentage of TTR editing in the liver after dosing LNPs formulated with mRNAs capped with the indicated transcription sequence at the indicated doses.
[00136] [00136] FIGURE 32 shows serum levels of TTR after dosing of LNPs formulated with mRNAs with the indicated cap and transcription sequence in the indicated amounts.
[00137] [00137] FIGURE 33 shows the percentage of TTR editing in the liver following dosing of LNPs formulated with mRNAs encoding Cas9 where the ORFs had the sequence of SEQ ID NO indicated, including an NLS as indicated.
[00138] [00138] Figures 34A-B show serum levels of TTR (A) and serum TTR (%TSS) (B) after dosing of LNPs formulated with mRNAs encoding Cas9 in which the ORFs had the sequence of SEQ ID NO indicated, including an NLS as indicated.
[00139] [00139] FIGURE 35 shows the correlation of NLS activity and editing efficiency after dosing of LNPs formulated with mRNAs encoding Cas9 and including NLS sequences of various classes and levels of activity.
[00140] [00140] FIGURE 36 shows Cas9 protein expression levels in HepG2 cells from mRNA transcripts with the indicated sequences and 5' UTRs as indicated. DETAILED DESCRIPTION OF CERTAIN MODALITIES
[00141] [00141] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that it is not intended to limit the invention to the embodiments. On the contrary, the invention is intended to encompass all alternatives, modifications and equivalents, which may be included within the invention as defined by the appended claims.
[00142] [00142] Before describing the present teachings in detail, it should be understood that the disclosure is not limited to specific compositions or process steps as they may vary. It should be noted that, as used herein in this specification, the singular forms "a", "an", and "the", "a" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates and reference to "a cell" includes a plurality of cells and the like.
[00143] [00143] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Furthermore, the use of "comprises", "comprises", "comprising", "contains", "contains", "containing", "includes", "includes" and "including" is not intended to be limiting. It is to be understood that the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.
[00144] [00144] The term "about" or "approximately" means an acceptable error for a specific value, as determined by one skilled in the art, which depends in part on how the value is measured or determined, or a degree of variation that is not substantially affects the properties of the described object, for example, within 10%, 5%, 2% or 1%. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations which may vary depending on the desired properties sought to be obtained. In the end, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of significant digits reported and by the application of ordinary rounding techniques.
[00145] [00145] Unless specifically stated in the specification above, modalities in the specification that recite "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components; modalities in the specification that recite "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the recited components; and modalities in the specification that recite "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of those terms in the claims).
[00146] [00146] Section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject in any way. In the event that any literature incorporated by reference contradicts the express content of this specification, including but not limited to a definition, the express content of this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. Rather, the present teachings encompass various alternatives, modifications and equivalents, as will be appreciated by those skilled in the art. A. Definitions
[00147] [00147] Unless otherwise noted, the following terms and phrases as used herein are intended to have the following meanings:
[00148] [00148] The term "or combinations thereof", as used herein, refers to all permutations and combinations of the listed terms that precede the term. For example, "A, B, C or combinations thereof" must include at least one of: A, B, C, AB, AC, BC or
[00149] [00149] As used herein, the term "kit" refers to a packaged set of related components such as one or more polynucleotides or compositions and one or more related materials such as delivery devices (e.g. syringes), solvents , solutions, buffers, instructions, or desiccants.
[00150] [00150] "Or" is used in the inclusive sense, that is, equivalent to "and/or", unless the context requires otherwise.
[00151] [00151] "Polynucleotide" and "nucleic acid" are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogues that have nitrogenous heterocyclic bases or analogues of bases linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA and polymers that are analogs thereof. A nucleic acid "backbone" can be made up of a variety of bonds, including one or more sugar-phosphodiester bonds, peptide-nucleic acid bonds ("peptide nucleic acids" or PNA; PCT WO 95/32305), phosphorothioate bonds , methylphosphonate bonds or combinations thereof. The sugar moieties of a nucleic acid can be ribose, deoxyribose or similar compounds with substitutions, for example, 2' methoxy or 2' halide substitutions. The nitrogenous bases can be conventional bases (A, G, C, T, U), analogues thereof (e.g. modified uridines such as 5-
[00152] [00152] "Modified uridine" is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, a modified uridine is a substituted uridine, that is, a uridine in which one or more non-proton substituents (eg, alkoxy, such as methoxy) take the place of a proton. In some embodiments, a modified uridine is pseudouridine. In some embodiments, a modified uridine is a substituted pseudouridine, that is, a pseudouridine in which one or more non-proton substituents (eg, alkyl, such as methyl) take the place of a proton. In some embodiments, a modified uridine is a substituted uridine, pseudouridine, or substituted pseudouridine.
[00153] [00153] "Uridine position" as used herein refers to a position in a polynucleotide occupied by a uridine or a modified uridine. So, for example, a polynucleotide in which "100% of the uridine positions are modified uridines" contains a modified uridine in all positions that would be uridines in a conventional RNA (where all the bases are standard A, U, C or G bases). ) of the same sequence. Unless otherwise indicated, a U in a polynucleotide sequence of or accompanying a sequence table or sequence listing, this disclosure may be a uridine or a modified uridine.
[00154] [00154] As used herein, a first sequence is considered "comprising a sequence with at least X% identity to" a second sequence if an alignment of the first sequence with the second sequence shows that X% or more of the positions of the second sequence in its entirety is matched by the first sequence. For example, the AAGA sequence comprises a sequence with 100% identity to the AAG sequence because an alignment would give 100% identity as there are matches for all three positions of the second sequence. Differences between RNA and DNA (usually the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogues such as modified uridines do not contribute to differences in identity or complementarity between polynucleotides, as long as the relevant nucleotides (such as thymidine , uridine or modified uridine) have the same complement (e.g. adenosine for all thymidine, uridine or modified uridine; another example is cytosine and 5-methylcytosine, both with guanosine as complement). So, for example, the 5'-AXG sequence where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG, as both are perfectly complementary to the same sequence (5' -CAU). Examples of alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One skilled in the art will understand which choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and identity and expected >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings from the Needleman-Wunsch algorithm interface provided by the EBI on the www.ebi.ac.uk web server is generally appropriate.
[00155] [00155] "mRNA" is used here to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino tRNAs -acylated). The mRNA may comprise a phosphate-sugar backbone including ribose residues or analogs thereof, for example 2'-methoxy ribose residues. In some embodiments, the sugars in a phosphate-sugar mRNA backbone consist essentially of ribose residues, 2'-methoxy-ribose residues, or a combination thereof. In general, mRNAs do not contain a substantial amount of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%,
[00156] [00156] As used herein, an "RNA-guided DNA binding agent" means a polypeptide or complex of polypeptides with RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the activity of DNA binding is sequence-specific and depends on the RNA sequence. Exemplary RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof ("dCas DNA binding agents"). "Cas nuclease", also called "Cas protein", as used herein, encompasses DNA binding agents of Cas cleavases, Cas nickases, and dCas DNA. DNA binding agents of Cas clevases/nickases include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1 or Cmr2 subunit, a cascade complex of a type I CRISPR system, the Cas3 subunit thereof and the Class 2 Cas nucleases. As used herein, a "Class 2 Cas nuclease" is a single-stranded polypeptide with RNA-guided DNA binding activity, such as a Cas9 nuclease or a Cpf1 nuclease. Class 2 Cas nucleases include Class 2 Cas clevases and Class 2 Cas nickases (e.g., variants H840A, D10A, or N863A), which further have RNA-guided DNA cleavages or nickase activity and Class 2 dCl binding agents, in which clevase/nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g. variants N497A, R661A, Q695A, Q926A), HypaCas9 (e.g. variants N692A, M694A, Q695A, H698A) , eSPCas9 1.0) (e.g. K810A, K1003A, R1060A variants) and eSPCas9 (1.1) (e.g. K848A, K1003A, R1060A variants) and modifications thereof. The Cpf1 protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. The Zetsche Cpf1 sequences are incorporated by reference in their entirety. See, for example, Zetsche, Tables S1 and S3. "Cas9" includes Spy Cas9, the variants of Cas9 listed here and equivalents thereof. See, for example, Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015 ); Shmakov et al., Molecular Cell, 60:385-397 (2015 ).
[00157] [00157] As used herein, the "minimum uridine content" of a given open reading frame (ORF) is the uridine toer of an ORF that (a) uses a minimum uridine codon at all positions and (b) ) encodes the same amino acid sequence as the given ORF. The uridine minimum codon(s) for a given amino acid is the codon(s) with the fewest uridines (usually 0 or 1, except a codon for phenylalanine, where the uridine minimum codon has 2 uridines). Modified uridine residues are considered equivalent to uridines in order to assess the minimum uridine content.
[00158] [00158] As used herein, the "minimum uridine dinucleotide content" of a given open reading frame (ORF) is the smallest possible uridine dinucleotide (UU) toer of an ORF that (a) uses a minimal codon of uridine (as discussed above) at all positions and (b) encodes the same amino acid sequence as the ORF provided. The uridine dinucleotide (UU) content can be expressed in absolute terms as the enumeration of UU dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by the uridines of uridine dinucleotides (e.g. AUUAU would have a 40% uridine dinucleotide content because 2 of 5 positions are occupied by the uridines of a uridine dinucleotide). Modified uridine residues are considered equivalent to uridines in order to assess the minimum uridine dinucleotide content.
[00159] [00159] As used in this document, the "minimum adenine content" of a given open reading frame (ORF) is the adenine toer of an ORF that (a) uses an adenine minimum codon at all positions and (b ) encodes the same amino acid sequence as the given ORF. The adenine min codon(s) for a given amino acid is the codon(s) with the least adenines (usually 0 or 1, except a codon for lysine and asparagine, where the adenine min codon has 2 adenines ). Modified adenine residues are considered equivalent to adenines in order to assess the minimum adenine content.
[00160] [00160] As used herein, the "minimum adenine dinucleotide content" of a given open reading frame (ORF) is the lowest possible adenine dinucleotide (AA) content of an ORF that (a) uses a minimum codon of adenine (as discussed above) at all positions and (b) encodes the same amino acid sequence as the ORF provided. Adenine dinucleotide (AA) content can be expressed in absolute terms as the enumeration of AA dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by adenines of adenine dinucleotides (e.g. UAAUA would have an adenine dinucleotide content of 40% because 2 of 5 positions are occupied by the adenines of an adenine dinucleotide). Modified adenine residues are considered equivalent to adenines in order to assess the minimum adenine dinucleotide content.
[00161] [00161] "Guide RNA", "gRNA", and "guide" are used interchangeably here to refer to a crRNA (also known as CRISPR RNA) or the combination of a crRNA and a trRNA (also known as tracrRNA). crRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (double guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA can be a naturally occurring sequence or a trRNA sequence with modifications or variations compared to naturally occurring sequences.
[00162] [00162] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. A "guide sequence" may also be referred to as a "target sequence" or "spacer sequence". A guide sequence may be 20 base pairs in length, for example in the case of Streptococcus pyogenes (e.g. Spy Cas9) and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used as guides, for example 15, 16, 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the target sequence is in a gene or chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence can be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100%. In some embodiments, the guide sequence and target region may be 100% complementary or identical. In other embodiments, the leader sequence and target region may contain at least one mismatch. For example, the leader sequence and the target sequence may contain 1, 2, 3 or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the leader sequence and target region may contain 1-4 mismatches, where the leader sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the leader sequence and target region may contain 1, 2, 3 or 4 mismatches, where the leader sequence comprises 20 nucleotides.
[00163] [00163] The target sequences of Cas proteins include the positive and negative strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence), as a nucleic acid substrate for a Cas protein is a double-stranded nucleic acid . Therefore, when a leader sequence is said to be "complementary to a target sequence", it should be understood that the leader sequence can direct a leader RNA to bind the reverse complement of a target sequence. Thus, in some embodiments, where the leader sequence binds the reverse complement of a target sequence, the leader sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence that does not include PAM), except for the replacement of U with T in the guide sequence.
[00164] [00164] As used herein, "indels" refers to insertion/deletion mutations that consist of multiple nucleotides that are inserted or deleted at the site of double-strand breaks (DSBs) in the target nucleic acid.
[00165] [00165] As used herein, "knockdown" refers to a decrease in the expression of a specific gene product (e.g., protein, mRNA, or both). Knockdown of a protein can be measured by detecting the protein secreted by the tissue or cell population (e.g., in serum or cell medium) or by detecting the total cellular amount of the protein from a tissue or cell population of interest. Methods for measuring mRNA knockdown are known and include sequencing mRNA isolated from a tissue or cell population of interest. In some embodiments, "knockdown" may refer to some loss of expression of a specific gene product, for example, a decrease in the amount of mRNA transcribed or a decrease in the amount of protein expressed or secreted by a population of cells (including inbred populations). alive as those found in tissues).
[00166] [00166] As used herein, "knockout" refers to a loss of expression of a specific protein in a cell. Knockout can be measured by detecting the amount of protein secretion from a tissue or cell population (e.g., in serum or cell medium) or by detecting the total cellular amount of a protein from a tissue or cell population. In some embodiments, the methods of the disclosure "knockout" a target protein in one or more cells (e.g., in a population of cells including in vivo populations such as those found in tissues). In some embodiments, a knockout is not the formation of the mutated target protein, for example created by indels, but the complete loss of expression of the target protein in a cell.
[00167] [00167] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, nickase, or DNA binding agent. dCas DNA (eg Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA-binding agent, such as Cas9, to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or a nickase, the linkage may be followed by cleavage or cleavage.
[00168] [00168] As used herein, a "target sequence" refers to a nucleic acid sequence in a target gene that has complementarity with the gRNA leader sequence. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind and potentially cut or cleave (depending on the activity of the agent) within the target sequence.
[00169] [00169] As used herein, "treatment" refers to any administration or application of a therapeutic for a disease or disorder to a subject and includes inhibiting the disease, halting its development, alleviating one or more symptoms of the disease, curing the disease or prevent the recurrence of one or more symptoms of the disease. B. Polynucleotides and Exemplary Compositions
[00170] [00170] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content that ranges from its minimal uridine content to about 150% uridine. its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102% or 101% of its minimum uridine content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content equal to its minimum uridine content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content less than or equal to about 150% of its minimum uridine content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content less than or equal to about 145% of its minimum uridine content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content less than or equal to about 140% of its minimum uridine content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content less than or equal to about 135% of its minimum uridine content.
[00171] [00171] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine dinucleotide content that ranges from its minimum uridine dinucleotide content to 200 % of its minimum uridine content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 195%, 190%, 185%, 180%, 175%, 170%, 165%, 160%, 155%, 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102% or 101% of its minimum uridine dinucleotide content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine dinucleotide content equal to its minimum uridine dinucleotide content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine dinucleotide content less than or equal to about 200% of its minimum uridine dinucleotide content. uridine. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine dinucleotide content less than or equal to about 195% of its minimum uridine dinucleotide content. uridine. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine dinucleotide content less than or equal to about 190% of its minimum uridine dinucleotide content. uridine.
[00172] [00172] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent that comprises an open reading frame (ORF) with a uridine dinucleotide content that varies from its minimum uridine dinucleotide content up to a uridine dinucleotide content that is 90% or less of the maximum uridine dinucleotide content of a reference sequence encoding the same protein as the mRNA in question. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15%, 10% or 5% of the maximum uridine dinucleotide content of a reference sequence encoding the same protein as the mRNA in question.
[00173] [00173] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine trinucleotide content ranging from 0 uridine trinucleotides to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 uridine trinucleotides (where a larger amount of uridines counts as the number of unique segments of three uridines within it, e.g. a uridine tetranucleotide contains two uridine trinucleotides, one uridine pentanucleotide contains three uridine trinucleotides, etc.). In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine trinucleotide content ranging from 0% uridine trinucleotides to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5% or 2% of uridine, where the percentage content of uridine trinucleotides is calculated as the percentage of positions in a sequence that are occupied by uridines that are part of a uridine trinucleotide (or greater number of uridines), so the sequences UUUAAA and UUUUAAAAA have a 50% uridine trinucleotide content. For example, in some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 2%. For example, in some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 1.5%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 1%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.9%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.8%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.7%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.6%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.5%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.4%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.3%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.2%. In some embodiments, the ORF has a uridine trinucleotide content of less than or equal to 0.1%. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) that does not contain uridine trinucleotides.
[00174] [00174] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine trinucleotide content that varies from its minimum uridine trinucleotide content up to a uridine trinucleotide content that is 90% or less of the maximum uridine trinucleotide content of a reference sequence encoding the same protein as the mRNA in question. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15%, 10% or 5% of the maximum uridine trinucleotide content of a reference sequence encoding the same protein as the mRNA in question.
[00175] [00175] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with minimal nucleotide homopolymers, eg repetitive strands of the same nucleotides. For example, in some embodiments, by selecting a minimal uridine codon from the codons listed in Table 1, an mRNA is constructed by selecting minimal uridine codons that reduce the number and length of nucleotide homopolymers, for example, by selecting GCA instead from GCC to alanine or selecting GGA instead of GGG to glycine or selecting AAG instead of AAA to lysine.
[00176] [00176] A given ORF can be reduced in uridine content or uridine dinucleotide content or uridine trinucleotide content, for example, by using uridine minimum codons in a sufficient portion of the ORF. For example, an amino acid sequence for an RNA-guided DNA binding agent can be back-translated into an ORF sequence by converting amino acids into codons, where part or all of the ORF uses the exemplary minimal uridine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons in the ORF are codons listed in Table 1. Table 1. Exemplary uridine min codons Amino acid Uridine min codon A Alanine GCA or GCC or GCG G Glycine GGA or GGC or GGG V Valine GUC or GUA or GUG
[00177] [00177] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) consisting of a set of codons of which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are codons listed in Table 1.
[00178] [00178] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with an adenine content ranging from its minimal adenine content to about 150% adenine. its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102% or 101% of its minimum adenine content. In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with an adenine content equal to its minimum adenine content.
[00179] [00179] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with an adenine dinucleotide content that ranges from its minimal adenine content to 200% adenine. its minimum adenine dinucleotide content. In some embodiments, the uridine adenine content of the ORF is less than or equal to about 195%, 190%, 185%, 180%, 175%, 170%, 165%, 160%, 155%, 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%,
[00180] [00180] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with an adenine dinucleotide content that varies from its minimum adenine dinucleotide content up to a uridine dinucleotide content that is 90% or less of the maximum adenine dinucleotide content of a reference sequence encoding the same protein as the mRNA in question. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15%, 10% or 5% of the maximum adenine dinucleotide content of a reference sequence encoding the same protein as the mRNA in question.
[00181] [00181] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with an adenine trinucleotide content ranging from 0 adenine trinucleotides to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 adenine trinucleotides (where a greater amount of adenines counts as the number of unique segments of three adenines within it, e.g. a adenine tetranucleotide contains two adenine trinucleotides, one adenine pentanucleotide contains three adenine trinucleotides, etc.). In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine trinucleotide content ranging from 0% adenine trinucleotides to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5% or 2% of adenine, where the percent adenine trinucleotide content is calculated as the percentage of positions in a sequence that are occupied by adenine that are part of an adenine trinucleotide (or greater adenine number), so the sequences UUUAAA and UUUUAAAAA have a content of 50% adenine trinucleotide.
[00182] [00182] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with minimal nucleotide homopolymers, eg repetitive strands of the same nucleotides. For example, in some embodiments, by selecting a minimal adenine codon from the codons listed in Table 1, an mRNA is constructed by selecting minimal adenine codons that reduce the number and length of nucleotide homopolymers, for example, by selecting GCA instead of of GCC for alanine or selecting GGA instead of GGG for glycine or selecting AAG instead of AAA for lysine.
[00183] [00183] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent that comprises an open reading frame (ORF) with an adenine trinucleotide content that varies from its minimum adenine trinucleotide content up to a uridine trinucleotide content that is 90% or less of the maximum adenine trinucleotide content of a reference sequence encoding the same protein as the mRNA in question. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35 %, 30%, 25%, 20%, 15%, 10% or 5% of the maximum adenine trinucleotide content of a reference sequence encoding the same protein as the mRNA in question.
[00184] [00184] A given ORF can be reduced in adenine content or adenine dinucleotide content or adenine trinucleotide content, for example, by using minimal adenine codons in a sufficient portion of the ORF. For example, an amino acid sequence for an RNA-guided DNA binding agent can be back-translated into an ORF sequence by converting amino acids into codons, where part or all of the ORF uses the exemplary minimal adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons in the ORF are codons listed in Table 2. Table 2. Exemplary adenine minimum codons Amino acid Adenine minimum codon A Alanine GCU or GCC or GCG G Glycine GGU or GGC or GGG V Valine GUC or GUU or GUG D Aspartic acid GAC or GAU E Glutamic acid GAG I Isoleucine AUC or AUU T Threonine ACU or ACC or ACG N Asparagine AAC or AAU K Lysine AAG S Serine UCU or UCC or UCG R Arginine CGU or CGC or CGG L Leucine CUG or CUC or CUU P Proline CCG or CCU or CCC H Histidine CAC or CAU Q Glutamine CAG F Phenylalanine UUC or UUU Y Tyrosine UAC or UAU C Cysteine UGC or UGU W Tryptophan UGG M Methionine AUG
[00185] [00185] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) consisting of a set of codons of which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are codons listed in Table 2.
[00186] [00186] To the extent possible, any of the features described above regarding low adenine may be combined with any of the features described above regarding low uridine. For example, an mRNA can be provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) with a uridine content that ranges from its minimum uridine content to about 150% of its minimum content. of uridine (for example, an ORF uridine content is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103 %, 102%, or 101% of its minimum uridine content) and an adenine content that ranges from its minimum adenine content to about 150% of its minimum adenine content (e.g. less than or equal to about 145% , 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102% or 101% of its minimum adenine content). The same is true of uridine and adenine dinucleotides. Likewise, the content of uridine nucleotides and adenine dinucleotides in the ORF can be as stated above. Likewise, the content of uridine dinucleotides and adenine nucleotides in the ORF can be as stated above.
[00187] [00187] A given ORF can be reduced in uridine and adenine nucleotide and/or dinucleotide content, for example, by using uridine and adenine minimum codons in a sufficient portion of the ORF. For example, an amino acid sequence for an RNA-guided DNA binding agent can be back-translated into an ORF sequence by converting amino acids into codons, where part or all of the ORF uses the exemplary minimal adenine and uridine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons in the ORF are codons listed in Table 3. Table 3. Exemplary Uridine and Adenine Min Codons Amino Acid Uridine Min Codon A Alanine GCC or GCG G Glycine GGC or GGG V Valine GUC or GUG D Aspartic acid GAC E Glutamic acid GAG I Isoleucine AUC T Threonine ACC or ACG N Asparagine AAC K Lysine AAG S Serine AGC or UCC or UCG R Arginine CGC or CGG L Leucine CUG or CUC P Proline CCG or CCC H Histidine CAC Q Glutamine CAG F Phenylalanine UUC Y Tyrosine UAC C Cysteine UGC W Tryptophan UGG M Methionine AUG
[00188] [00188] In some embodiments, an mRNA is provided that encodes an RNA-guided DNA binding agent comprising an open reading frame (ORF) consisting of a set of codons of which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are codons listed in Table 3. As can be seen from Table 3, each of the three listed serine codons contains an A or a U. In In some modalities, uridine minimization is prioritized using AGC codons for serine. In some embodiments, adenine minimization is prioritized using the UCC and/or UCG codons for serine.
[00189] [00189] In some embodiments, the mRNA comprises an ORF with codons that enhance translation in a mammal, such as a human. In other embodiments, the mRNA comprises an ORF with codons that enhance translation in an organ, such as the liver, of the mammal, for example, a human. In other embodiments, the mRNA comprises an ORF with codons that enhance translation in a cell type, such as a mammalian hepatocyte, for example, a human. An increase in translation in a mammal, cell type, organ of a mammal, human, organ of a human, etc., can be determined with respect to the extent of translation of the wild-type sequence of the ORF or with respect to an ORF with a codon distribution corresponding to the codon distribution of the organism from which the ORF was derived or of the organism containing the most similar ORF at the amino acid level, such as S. pyogenes, S. aureus, other prokaryotes, as the case may be for Cas nucleases derived in prokaryotes, such as the Cas nucleases of other prokaryotes described below. Alternatively, in some embodiments, an increase in translation for a Cas9 sequence in a mammal, cell type, mammalian organ, human, human organ, etc., is determined with respect to translation of an ORF with the sequence of the SEQ ID NO: 5 with all others the same, including any applicable point mutations, heterologous domains and the like. Useful codons to increase expression in a human, including human liver and human hepatocytes, may be codons corresponding to highly expressed tRNAs in human liver/hepatocytes, discussed in Dittmar KA, PLos Genetics 2(12): e221 (2006) ). In some embodiments, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons that correspond to highly expressed tRNAs. (for example, the most expressive tRNA for each amino acid) in a mammal, such as a human. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons corresponding to highly expressed tRNAs (e.g. , the highest expressed tRNA for each amino acid) in a mammalian organ, such as a human organ. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons that correspond to highly expressed tRNAs (e.g. (e.g., the highest-expressing tRNA for each amino acid) in a mammalian liver, such as a human liver. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons corresponding to highly expressed tRNAs (e.g. , the highest expressed tRNA for each amino acid) in a mammalian hepatocyte, such as a human hepatocyte.
[00190] [00190] Alternatively, codons corresponding to tRNAs highly expressed in an organism (eg human) in general can be used.
[00191] [00191] Any of the above approaches to codon selection can be combined with the minimum uridine and/or adenine codons shown above, e.g. starting with codons from Table 1, 2 or 3 and then when further option is available, using the codon that corresponds to a more highly expressed tRNA, either in the organism (e.g., human) in general, or in an organ or cell type of interest, such as liver or hepatocytes
[00192] [00192] In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons in a codon set shown in Table 4 (for example, the low U1, A low, or A/U low codon set). Codons in the low U1, low G, low C, A, and low A/U sets use codons that minimize the indicated nucleotides while also using codons corresponding to highly expressed tRNAs where more than one option is available. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in an ORF are codons from the low U1 codon set shown in Table
[00193] [00193] In some embodiments, the RNA-guided DNA binding agent is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA binding agent has cleavase activity, which may also be referred to as endonuclease activity of double tape. In some embodiments, the RNA-guided DNA binding agent comprises a Cas nuclease, such as a Class 2 Cas nuclease (which can be, for example, a Type II, V, or VI Cas nuclease). Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2 and C2c3 proteins and modifications thereof. Examples of Cas9 nucleases include those from the type II CRISPR systems of S. pyogenes, S. aureus and other prokaryotes (see, for example, the list in the next paragraph) and modified versions (eg, engineered or mutant). See, for example, US2016/0312198 A1; US 2016/0312199 A1. Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1 or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system or its Cas3 subunit thereof. In some embodiments, the Cas nuclease may be of a Type IIA, Type IIB, or Type IIC system. For discussion of various CRISPR systems and Cas nucleases, see, for example, Makarova et al., NAT. REV. MICROBIOL. 9:467-477 (2011 ); Makarova et al., NAT. REV. MICROBIOL, 13: 722-36 (2015 ); Shmakov et al., MOLECULAR CELL, 60:385-397 (2015 ).
[00194] Exemplary non-limiting species from which Cas nuclease can be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, succinogene Fibrobacter, Rhodospirillum rubrum, Nocardiopsis dassonvillei, pristinaespiralis Streptomyces, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, salivarius Lactobacillus, Lactobacillus buchneri , Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synech ococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrobacter sp. Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthanoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Occupis pasteurianus, Streptoc. cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina.
[00195] [00195] In some embodiments, the Cas nuclease is Streptococcus pyogenes Cas9 nuclease. In some embodiments, the Cas nuclease is Streptococcus thermophilus Cas9 nuclease. In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is Staphylococcus aureus Cas9 nuclease. In some embodiments, the Cas nuclease is Francisella novicida Cpf1 nuclease. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In other embodiments, the Cas nuclease is Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.
[00196] [00196] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target strand of DNA, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild-type Cas9. In some embodiments, Cas9 is able to induce a double-strand break in the target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, may cleave a strand of dsDNA, or may lack DNA clevase or nickase activity. An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 3. An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 4. An exemplary Cas9 mRNA coding sequence , suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 10.
[00197] [00197] In some embodiments, chimeric Cas nucleases are used, in which a domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease, such as Fok1. In some embodiments, a Cas nuclease can be a modified nuclease.
[00198] [00198] In other embodiments, the Cas nuclease may be from a CRISPR/Cas Type I system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a CRISPR/Cas Type I system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a CRISPR/Cas Type I system. Cas nuclease can be a Cas3 protein. In some embodiments, the Cas nuclease may be from a CRISPR/Cas Type III system. In some embodiments, the Cas nuclease may have RNA-cleaving activity.
[00199] [00199] In some embodiments, the RNA-guided DNA binding agent has single-stranded nickase activity, i.e., it can cut a strand of DNA to produce a single-strand break, also known as a "cut". In some embodiments, the RNA-guided DNA binding agent comprises a Cas nickase. A nickase is an enzyme that creates a cut in dsDNA, that is, cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more changes (e.g., point mutations) in a catalytic domain. See, for example, US Patent 8,889,356 for discussion of Cas nickases and exemplary changes in the catalytic domain. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. An exemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO: 6. An exemplary Cas9 nickase mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 7. An mRNA coding sequence of Exemplary Cas9 nickase, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 11.
[00200] [00200] In some embodiments, the RNA-guided DNA binding agent is modified to contain only a functional nuclease domain. For example, the agent protein can be modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleaving activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.
[00201] [00201] In some embodiments, a conserved amino acid within a nuclease domain of the Cas protein is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC nuclease or RuvC-like domain. Exemplary amino acid substitutions in the RuvC nuclease or RuvC-like domain include D10A (based on the Cas9 protein from S. pyogenes). See, for example, Zetsche et al. (2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH nuclease or HNH-like domain. Exemplary amino acid substitutions in the HNH nuclease or HNH-like domain include E762A, H840A, N863A, H983A and D986A (based on the Cas9 protein of S. pyogenes). See, for example, Zetsche et al. (2015). Other exemplary amino acid substitutions include D917A, E1006A and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB - A0Q7Q2 (CPF1_FRATN)).
[00202] [00202] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, guide RNAs direct the nickase to a target sequence and introduce a DSB generating a nick on opposite strands of the target sequence (i.e., double snip). In some modalities, the use of double cut can improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs, directing opposite strands of DNA, to produce a double cut in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be close together to produce a double cut in the target DNA.
[00203] [00203] In some embodiments, the RNA-guided DNA binding agent lacks clevase and nickase activity. In some embodiments, the RNA-guided DNA binding agent comprises a dCas DNA binding polypeptide. A dCas polypeptide has DNA binding activity, while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking clevase and nickase activity or the dCas-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, for example, by one or more changes (e.g. point mutations) in their catalytic domains. See, for example, US 2014/0186958 A1; US 2015/0166980 A1. An exemplary dCas9 amino acid sequence is provided as SEQ ID NO: 8. An exemplary dCas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 9. An exemplary dCas9 mRNA coding sequence , suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 12.
[00204] [00204] In some embodiments, the RNA-guided DNA binding agent comprises one or more heterologous functional domains (eg, is or comprises a fusion polypeptide).
[00205] [00205] In some embodiments, the heterologous functional domain can facilitate RNA-guided transport of the DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binding agent can be fused to 1-10 NLS(s). In some embodiments, the RNA-guided DNA binding agent can be fused to 1-5 NLS(s). In some embodiments, the RNA-guided DNA binding agent may be fused to an NLS. When an NLS is used, the NLS can be ligated at the N-terminus or C-terminus of the RNA-guided DNA-binding agent sequence. In some embodiments, the RNA-guided DNA binding agent may be fused at the C-terminus with at least one NLS. An NLS can also be inserted into the RNA-guided DNA binding agent sequence. In other embodiments, the RNA-guided DNA binding agent may be fused to more than one NLS. In some embodiments, the RNA-guided DNA binding agent can be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA binding agent can be fused with two NLSs. In certain circumstances, the two NLSs can be the same (for example, two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA binding agent can be fused to two NLSs, one N-terminal and one C-terminal. In some embodiments, the RNA-guided DNA binding agent can be fused with 3 NLSs. In some embodiments, the RNA-guided DNA binding agent may be fused to no NLS. In some embodiments, the NLS may be a single-part sequence, such as, for example, the SV40 NLS, PKKKRKV (SEQ ID NO: 78) or PKKKRRV (SEQ ID NO: 90). In some embodiments, the NLS may be a bipartite sequence, such as the nucleoplasmin NLS, KRPAATKKAGQAKKKK (SEQ ID NO: 91). In some embodiments, the NLS sequence may comprise LAAKRSRTT (SEQ ID NO: 79), QAAKRSRTT (SEQ ID NO: 80), PAPAKRERTT (SEQ ID NO: 81), QAAKRPRTT (SEQ ID NO: 82), RAAKRPRTT (SEQ ID NO: 82). : 83), AAAKRSWSMAA (SEQ ID NO: 84), AAAKRVWSMAF (SEQ ID NO: 85), AAAKRSWSMAF (SEQ ID NO: 86), AAAKRKYFAA (SEQ ID NO: 87), RAAKRKAFAA (SEQ ID NO: 88) or RAAKRKYFAV (SEQ ID NO: 89). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 78) NLS can be ligated at the C-terminus of the RNA-guided DNA binding agent. One or more linkers are optionally included at the fusion site. In some embodiments, one or more NLS(s) according to any of the foregoing embodiments are present in the RNA-guided DNA binding agent in combination with one or more additional heterologous functional domains, such as any of the heterologous functional domains described below. .
[00206] [00206] In some embodiments, the heterologous functional domain may be able to modify the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be shortened. In some embodiments, the heterologous functional domain may be able to enhance the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain may be able to reduce the stability of the RNA-guided DNA binding agent. In some embodiments, the heterologous functional domain can act as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binding agent can be modified by the addition of ubiquitin or a chain of polubiquitin. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-15-stimulated gene 15 (ISG15)), ubiquitin-related modifier-1 (URM1) , developmentally dysregulated protein-8 expressed in neuron precursor cells (NEDD8, also called Rub1 in S. cerevisiae), associated with human leukocyte antigen F (FAT10), autophagy-8 (ATG8) and -12 (ATG12), similar protein the Fau ubiquitin (FUB1), membrane-anchored UBL (MUB), ubiquitin-fold modifier-1 (UFM1) and ubiquitin-like protein-5 (UBL5).
[00207] [00207] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of tag domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g. e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, Sapphire-T), cyan fluorescent proteins (e.g. ECFP, cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g. mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRed1, AsRed2, eRedberry2 , mStrawberry, Jred) and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Komabira-Monomeric-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the tag domain can be a purification tag and/or an epitope tag. Exemplary non-limiting tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly (NANP), tandem affinity purification tag ( TAP), myc, AcV5 , AU1, AU5, E, ECS, E2, FLAG, HA, nudes, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV -G, 6xHis, 8xHis, biotin carboxyl transporter protein (BCCP), poly-His and calmodulin. Exemplary non-limiting reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
[00208] [00208] In additional embodiments, the heterologous functional domain can target the RNA-guided DNA binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain can direct the RNA-guided DNA binding agent to mitochondria.
[00209] [00209] In other embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA binding agent is targeted to its target sequence, for example, when a Cas nuclease is targeted to a target sequence by a gRNA, the effector domain can modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a repressor domain. transcriptional. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, for example, US Patent 9,023,649. In some embodiments, the heterologous functional domain is an activator or repressor of transcription. See, for example, Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nicknames for cooperative genome engineering,” Nat. Biotechnol. 31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51 (2013). As such, the RNA-guided DNA binding agent essentially becomes a transcription factor that can be targeted to bind a desired target sequence using a guide RNA. In certain embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain, such as a base editing domain. In particular embodiments, the DNA modification domain is a nucleic acid editing domain that introduces a specific modification into the DNA, such as a deaminase domain. See, for example, WO 2015/089406; US 2016/0304846. The nucleic acid editing domains, deaminase domains and Cas9 variants described in WO 2015/089406 and US 2016/0304846 are incorporated herein by reference.
[00210] [00210] In some embodiments, the mRNA comprises at least one UTR of hydroxysteroid 17-beta dehydrogenase 4 (HSD17B4 or HSD), for example, a 5' UTR of HSD. In some embodiments, the mRNA comprises at least one UTR of a globin mRNA, for example, human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In some embodiments, the mRNA comprises a 5' UTR, 3' UTR, or 5' and 3' UTRs of a globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5' UTR of bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3' UTR of bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises 5' and 3' UTRs of bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53) or epidermal growth factor receptor (EGFR).
[00211] [00211] In some embodiments, the mRNA comprises 5' and 3' UTRs that are from the same source, for example, an mRNA constitutively expressed as actin, albumin or globin as HBA, HBB or XBG.
[00212] [00212] In some embodiments, an mRNA disclosed herein comprises a 5' UTR with at least 90% identity to any of SEQ ID NOs: 32, 34, 36, 38, or 41. In some embodiments, an mRNA disclosed herein document comprises a 3' UTR with at least 90% identity to any one of SEQ ID NOs: 33, 35, 37, 39, or 40. In some embodiments, any of the foregoing levels of identity is at least 95%, at least least 98%, at least 99% or 100%. In some embodiments, an mRNA disclosed herein comprises a 5' UTR with the sequence of any one of SEQ ID NOs: 32, 34, 36, 38, or 41. In some embodiments, an mRNA disclosed herein comprises a 3' UTR with the sequence of any one of SEQ ID NOs: 33, 35, 37, 39 or 40.
[00213] [00213] In some embodiments, the mRNA does not comprise a 5' UTR, for example, there are no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA comprises a Kozak sequence (described below) between the 5' cap and the start codon, but has no additional 5' UTR. In some embodiments, the mRNA does not comprise a 3' UTR, for example, there are no additional nucleotides between the stop codon and the poly-A tail.
[00214] [00214] In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect the initiation of translation and the overall yield of a polypeptide translated from an mRNA. A Kozak sequence includes a methionine codon that can function as the start codon. A minimal Kozak sequence is NNNRUGN where at least one of the following is true: the first N is A or G and the second N is G. In the context of a nucleotide sequence, R means a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero mismatches or with up to one or two mismatches for lowercase positions. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or with up to one or two mismatches for lowercase positions. In some embodiments, the Kozak sequence is gccRccAUGG (nucleotides 4 to 13 of SEQ ID NO: 105) with zero mismatches or with up to one, two or three mismatches for lowercase positions. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or with up to one, two, three, or four mismatches for lowercase positions. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG (SEQ ID NO: 105) with zero mismatches or with up to one, two, three, or four mismatches for lowercase positions.
[00215] [00215] In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binding agent, wherein the ORF comprises a sequence with at least 90% identity to any of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175. In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent comprises an amino acid sequence with at least 90% identity to any of the SEQ IDs NOs: 3, 6, 8, 13, 16, 19, 22, 25, 28, 68, or 186-196, where the ORF has a uridine content that ranges from its minimum uridine content to 150% of its minimum uridine content. uridine and/or has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content. In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binding agent, wherein the RNA-guided DNA binding agent comprises an amino acid sequence with at least 90% identity to any of the SEQ IDs NOs: 3, 6, 8, 13, 16, 19, 22, 25, 28, 68, or 186-196, where the ORF has an adenine content that ranges from its minimum adenine content to 150% of the minimum adenine content. adenine and/or has an adenine dinucleotide content ranging from its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content. In some of these embodiments, the content of adenine and uridine nucleotides is less than or equal to 150% of their respective minima. In some embodiments, the content of adenine and uridine dinucleotide is less than or equal to 150% of their respective minima. In some embodiments, the mRNA comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 67, wherein the sequence comprises an ORF encoding an agent of binding to DNA guided by RNA. In some embodiments, the mRNA comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 67, wherein the sequence comprises an ORF encoding a guided DNA by RNA linker, wherein the first three nucleotides of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 67 are omitted. In some embodiments, any of the above identity levels is at least 95%, at least 98%, at least 99%, or 100%.
[00216] [00216] In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binding agent, wherein the ORF has at least 90% identity to any of the SEQs
[00217] [00217] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 43, optionally wherein the ORF of SEQ ID NO: 43 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00218] [00218] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 44, optionally wherein the ORF of SEQ ID NO: 44 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00219] [00219] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 56, optionally wherein the ORF of SEQ ID NO: 56 (i.e. SEQ ID NO: 4) is replaced by an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00220] [00220] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 57, optionally wherein the ORF of SEQ ID NO: 57 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00221] [00221] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 58, optionally wherein the ORF of SEQ ID NO: 58 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00222] [00222] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 59, optionally wherein the ORF of SEQ ID NO: 59 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00223] [00223] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 60, optionally wherein the ORF of SEQ ID NO: 60 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00224] [00224] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 61, optionally wherein the ORF of SEQ ID NO: 61 (i.e. SEQ ID NO: 4) is replaced with an alternate ORF of any one of SEQ ID NOs: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23 , 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
[00225] [00225] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 176, optionally wherein the ORF of SEQ ID NO: 176 is replaced with an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00226] [00226] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 177, optionally wherein the ORF of SEQ ID NO: 177 is replaced with an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00227] [00227] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 178, optionally wherein the ORF of SEQ ID NO: 178 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00228] [00228] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 179, optionally wherein the ORF of SEQ ID NO: 179 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00229] [00229] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 180, optionally wherein the ORF of SEQ ID NO: 180 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00230] [00230] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 181, optionally wherein the ORF of SEQ ID NO: 181 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00231] [00231] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 182, optionally wherein the ORF of SEQ ID NO: 182 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52,
[00232] [00232] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 183, optionally wherein the ORF of SEQ ID NO: 183 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00233] [00233] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 184, optionally wherein the ORF of SEQ ID NO: 184 is replaced by an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00234] [00234] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 185, optionally wherein the ORF of SEQ ID NO: 185 is replaced with an alternate ORF of any one of SEQ ID NO: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 , 30, 50, 52, 54, 65, 66 or 107-175.
[00235] [00235] In some embodiments, the degree of identity for the optionally substituted sequences of SEQ ID NOs 43, 44, 56-61 or 176-185 is at least 95%. In some embodiments, the degree of identity for the optionally substituted sequences of SEQ ID NOs 43, 44, 56-61, or 176-185 is at least 98%. In some embodiments, the degree of identity for the optionally substituted sequences of SEQ ID NOs 43, 44, 56-61, or 176-185 is at least 99%. In some embodiments, the degree of identity for the optionally substituted sequences of SEQ ID NOs 43, 44, 56-61, or 176-185 is 100%.
[00236] [00236] In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail. In some cases, the poly-A tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more sites within the poly-A tail. Poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotides. As used herein, "non-adenine nucleotides" refers to natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tails in the mRNA described herein may comprise consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or sequence of interest. In some cases, poly-A tails in mRNA comprise non-consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA binding agent or sequence of interest, where non-adenine nucleotides interrupt adenine nucleotides at intervals regularly or irregularly spaced.
[00237] [00237] In some embodiments, the poly-A tail is encoded on the plasmid used for in vitro transcription of mRNA and becomes part of the transcript. The poly-A sequence encoded in the plasmid, i.e. the number of consecutive adenine nucleotides in the poly-A sequence, may not be exact, e.g. a sequence of 100 poly-A in the plasmid may not precisely result in a sequence of 100 poly-A in the transcribed mRNA. In some embodiments, the poly-A tag is not encoded on the plasmid and is added by PCR tag or enzymatic tag, for example, using E. coli poly(A) polymerase.
[00238] [00238] In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt consecutive adenine nucleotides so that a poly(A) binding protein can bind a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8-100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotide is after one, two, three, four, five, six or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides.
[00239] [00239] The poly-A tail of the present disclosure may comprise a sequence of consecutive adenine nucleotides followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.
[00240] [00240] In some embodiments, the poly-A tail comprises or contains a non-adenine nucleotide or a consecutive stretch of 2-10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide(s) are located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some cases, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.
[00241] [00241] In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some cases, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some cases, where more than one non-adenine nucleotide is present, the non-adenine nucleotide can be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An example of a poly-A tail comprising non-adenine nucleotides is provided as SEQ ID NO: 62.
[00242] [00242] In some embodiments, an mRNA comprises a uridine modified at some or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, for example, with a halogen or C1-C3 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, for example, with a C1-C3 alkyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.
[00243] [00243] In some modalities, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95%, 98%, 99% or 100% of the uridine positions in an mRNA according to the disclosure are modified uridines. In some modes, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are modified uridines, for example 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine, pseudouridine or a combination thereof. In some modes, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-methoxyuridine. In some modes, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are pseudouridine. In some modes, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are N1-methyl pseudouridine. In some modes, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine. In some modes, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-methoxyuridine and the remainder is N1-methyl pseudouridine. In some modes, 10% - 25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine and the remainder is N1-methyl pseudouridine.
[00244] [00244] In some embodiments, an mRNA disclosed herein comprises a 5' cap, such as a Cap0, Cap1, or Cap2. A 5' cap is usually a 7-methylguanine ribonucleotide (which may be further modified, as discussed below, for example with respect to ARCA) linked via a 5'-triphosphate to the 5' position of the first nucleotide of the 5' chain. -a-3' of the mRNA, that is, the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA comprise a 2'-hydroxyl. In Cap1, the riboses of the first and second nucleotides transcribed from the mRNA comprise a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA comprise a 2'-methoxy. See, for example, Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30 ; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most higher endogenous eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2. Cap0 and capping structures other than Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, which can result in levels levels of cytokines, including type I interferon. Components of the innate immune system, such as IFIT-1 and IFIT-5, may also compete with eIF4E for binding an mRNA with a capping different from Cap1 or Cap2, potentially inhibiting the mRNA translation.
[00245] [00245] A cap may be included co-transcriptionally. For example, ARCA (anti-reverse capping analog; Thermo Fisher Scientific Cat. No. AM8045) is a capping analog comprising a 7-methylguanine 3'-methoxy-5'-triphosphate linked at the
[00246] [00246] CleanCapTM AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCapTM GG (m7G(5')ppp(5')(2' OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally. The 3'-O-methylated versions of CleanCapTM AG and CleanCapTM GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively. The CleanCapTM AG structure is shown below. CleanCapTM structures are sometimes referred to here using the last three digits of the catalog numbers listed above (eg, “CleanCapTM 113” for TriLink Biotechnologies Cat. No. N-7113).
[00247] [00247] Alternatively, a cap can be added to a
[00248] [00248] In some embodiments, at least one guide RNA is provided in combination with an mRNA disclosed herein. In some embodiments, a guide RNA is provided as a separate molecule from the mRNA. In some embodiments, a guide RNA is provided as a part, as a part of a UTR, of an mRNA disclosed herein. In some embodiments, at least one guide RNA directs TTR.
[00249] [00249] In some embodiments, a guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in SEQ ID NO: 74, where N is any natural or unnatural nucleotide and where all of the N's comprise a leader sequence. For example, included herein is SEQ ID NO: 74, where the Ns are replaced by any of the guide sequences disclosed herein. The modifications are shown in SEQ ID NO: 74, despite the replacement of N by the nucleotides of a guide. That is, although the guide nucleotides replace the “Ns”, the first three nucleotides are modified in 2'OMe and there are phosphorothioate bonds between the first and second nucleotides, the second and third nucleotides and the third and fourth nucleotides.
[00250] [00250] In some embodiments, an mRNA described herein, alone or accompanied by one or more guide RNAs, is formulated or delivered via a lipid nanoparticle; see, for example, PCT/US2017/024973, filed March 30, 2017, claiming priority to USSN 62/315,602, filed March 30, 2016 and titled “LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS COMPONENTS,” the contents of which are incorporated herein by reference in its entirety. Any lipid nanoparticle (LNP) known to those skilled in the art to be capable of delivering nucleotides to subjects can be used to deliver the RNAs described herein, which in some embodiments are accompanied by one or more guide RNAs. In some embodiments, an mRNA described herein, alone or accompanied by one or more guide RNAs, is formulated or administered via a liposome, a nanoparticle, an exosome, or a microvesicle. Emulsions, micelles and suspensions may be compositions suitable for local and/or topical delivery.
[00251] [00251] Disclosed herein are various modalities of LNP formulations for RNAs, including CRISPR/Cas payloads. Such LNP formulations may include (i) a CCD lipid, such as an amine lipid, (ii) a neutral lipid, (iii) an auxiliary lipid, and (iv) a stealth lipid, such as a PEG lipid. Some embodiments of LNP formulations include an "amine lipid", along with an auxiliary lipid, a neutral lipid, and a stealth lipid, such as a PEG lipid. By "lipid nanoparticle" is meant a particle comprising a plurality of (i.e., more than one) lipid molecules physically associated with each other by intermolecular forces. CCD lipids
[00252] [00252] Lipid compositions for delivering CRISPR/Cas mRNA and guide RNA components to a liver cell comprise a CCD lipid.
[00253] [00253] In some embodiments, the lipid of the CCD is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(( diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy) )carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be described as: .
[00254] [00254] Lipid A can be synthesized according to WO2015/095340 (eg pp. 84-86).
[00255] [00255] In some embodiments, the CCD lipid is lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-di-yl )bis(decanoate), also called ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-di-yl)bis(decanoate). Lipid B can be described as: .
[00256] [00256] Lipid B can be synthesized according to WO2014/136086 (eg pp. 107-09).
[00257] [00257] In some embodiments, the CCD lipid is lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-di-yl (9Z,9'Z,12Z, 12'Z)-bis(octadeca-9,12-dienoate).
[00258] [00258] Lipid C can be described as:
[00259] [00259] In some embodiments, the CCD lipid is lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate.
[00260] [00260] Lipid D can be described as: .
[00261] [00261] Lipid C and lipid D can be synthesized according to WO2015/095340.
[00262] [00262] CCD lipid may also be equivalent to lipid A, lipid B, lipid C, or lipid D. In certain embodiments, the CCD lipid is equivalent to lipid A, a lipid B equivalent, a lipid C equivalent, or an equivalent to lipid D.
[00263] [00263] Amine lipids
[00264] [00264] In some embodiments, LNP compositions for the delivery of biologically active agents comprise an "amine lipid", which is defined as lipid A or its equivalents, including acetal analogues of lipid A.
[00265] [00265] In some embodiments, the amine lipid is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-
[00266] [00266] Lipid A can be synthesized according to WO2015/095340 (eg pp. 84-86). In certain embodiments, the amine lipid is equivalent to lipid A.
[00267] [00267] In certain embodiments, an amine lipid is a lipid A analog. In certain embodiments, a lipid A analog is a lipid A acetal analog. In certain LNP compositions, the acetal analog is a C4- acetal analog. C12. In some embodiments, the acetal analogue is a C5-C12 acetal analogue. In additional embodiments, the acetal analogue is a C5-C10 acetal analogue. In other embodiments, the acetal analogue is chosen from an acetal analogue of C4, C5, C6, C7, C9, C10, C11 and C12.
[00268] [00268] Amine lipids suitable for use in the LNPs described herein are biodegradable in vivo. Amine lipids have low toxicity (eg, they are tolerated in animal models without adverse effects in amounts greater than or equal to 10 mg/kg). In certain embodiments, LNPs comprising an amine lipid include those in which at least 75% of the amine lipid is removed from the plasma within 8, 10, 12, 24 or 48 hours or 3, 4, 5, 6, 7 or 10 days. In certain embodiments, LNPs that comprise an amine lipid include those in which at least 50% of the mRNA or gRNA is cleared from plasma within 8, 10, 12, 24, or 48 hours or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising an amine lipid include those in which at least 50% of the LNP is cleared from plasma within 8, 10, 12, 24, or 48 hours or 3, 4, 5, 6, 7, or 10 days. , for example, measuring a lipid (eg, an amine lipid), RNA (eg, mRNA), or other component. In certain embodiments, the lipid-encapsulated component of the lipid versus free lipid, RNA, or nucleic acid of the LNP is measured.
[00269] [00269] Lipid clearance can be measured as described in the literature. See Maier, MA, et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Ther. 2013, 21(8), 1570-78 (“Maier”). For example, in Maier, LNP-siRNA systems containing luciferase-targeted siRNA were administered to six to eight week old male C57Bl/6 mice at 0.3 mg/kg by intravenous bolus injection via the lateral tail vein. Blood, liver and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96 and 168 hours post-dose. Mice were perfused with saline before tissue collection and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. In addition, Maier describes a procedure to assess toxicity after administration of LNP-siRNA formulations. For example, a luciferase-targeted siRNA was administered at 0, 1, 3, 5, and 10 mg/kg (5 animals/group) via intravenous bolus injection at a dose volume of 5 mL/kg in Sprague-Dawley rats. males. After 24 hours, about 1 ml of blood was obtained from the jugular vein of conscious animals and the serum was isolated. 72 hours post-dose, all animals were sacrificed for necropsy. Clinical signs, body weight, serum chemistry, organ weights and histopathology were evaluated. Although Maier describes methods for evaluating siRNA-LNP formulations, those methods can be applied to evaluate the clearance, pharmacokinetics, and toxicity of administering LNP compositions of the present disclosure.
[00270] [00270] Amine lipids lead to an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example, the rate at which an amine lipid is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example, the rate at which an mRNA or gRNA is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from a tissue, such as liver tissue or spleen tissue. In certain modalities, a high clearance rate leads to a safety profile without substantial adverse effects. Amine lipids reduce LNP accumulation in circulation and tissues. In some modalities, a reduction in LNP accumulation in the circulation and tissues leads to a safety profile without substantial adverse effects.
[00271] [00271] The amine lipids of the present disclosure may be ionizable depending on the pH of the medium they are in. For example, in a slightly acidic medium, amine lipids can be protonated and thus carry a positive charge. On the other hand, in a slightly basic medium, such as blood where the pH is approximately 7.35, amine lipids may not be protonated and therefore have no charge. In some embodiments, the amine lipids of the present disclosure can be protonated to a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure can be protonated to a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure can be protonated to a pH of at least about 9. embodiments, the amine lipids of the present disclosure can be protonated to a pH of at least about 10.
[00272] [00272] The ability of an amine lipid to bear a charge is related to its intrinsic pKa. For example, the amine lipids of the present disclosure can each independently have a pKa in the range of about 5.8 to about 6.2. For example, the amine lipids of the present disclosure can each independently have a pKa in the range of about 5.8 to about 6.5. This may be advantageous as it has been found that cationic lipids with a pKa ranging between 5.1 and 7.4 are effective for load distribution in vivo, for example to the liver. Furthermore, cationic lipids with a pKa ranging from about 5.3 to about 6.4 have been found to be effective for in vivo delivery, for example to tumors. See, for example, WO2014/136086. additional lipids
[00273] [00273] Suitable "neutral lipids" for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pophospholine (DOPC), dimyristoylphosphatidylcholine (DMPC) , phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilaurylylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2 -palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diaracidiloyl-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC) ), 1,2-dieicosenoyl-sn-glycero-3-phosphochololine (DEPC), palmitoylloylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl phosphatid acetylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).
[00274] [00274] "Helper lipids" include steroids, sterols and alkyl resorcinols. Auxiliary lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.
[00275] [00275] "Stealth lipids" are lipids that alter the length of time nanoparticles can exist in vivo (eg, in blood). Stealth lipids can assist in the formulation process, for example by reducing particle aggregation and controlling particle size. The stealth lipids used here can modulate the pharmacokinetic properties of LNP. Stealth lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, stealth lipids with a hydrophilic head group attached to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information on the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, for example, in WO 2006/007712.
[00276] [00276] In one embodiment, the stealth lipid hydrophilic head group comprises a polymer moiety selected from PEG-based polymers. Stealth lipids may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG
[00277] [00277] In one embodiment, a stealth lipid comprises a polymer moiety selected from PEG-based polymers (sometimes called poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids and poly[N-(2-hydroxypropyl)methacrylamide].
[00278] [00278] In one embodiment, the PEG lipid comprises a PEG-based polymer moiety (sometimes referred to as poly(ethylene oxide)).
[00279] [00279] The PEG lipid further comprises a lipid moiety. In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising an alkyl chain-length dialkylglycerol or dialkylglycamide group independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional carbon groups such as, for example, an amide or ester. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglycamide group may further comprise one or more substituted alkyl groups. Chain lengths can be symmetrical or asymmetrical.
[00280] [00280] Unless otherwise indicated, the term "PEG", as used herein, means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, the PEG is not replaced. In one embodiment, the PEG is substituted, for example, by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In one embodiment, the term includes PEG copolymers, such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry:
[00281] [00281] In certain embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid), is a "PEG-2K", also called "PEG 2000", which has an average molecular weight of about of 2,000 daltons. PEG-2K is represented herein by the following formula (I), where n is 45, meaning that the number average degree of polymerization comprises about 45 subunits. However, other PEG modalities known in the art can be used, including, for example, those where the average degree of polymerization comprises about 23 subunits (n=23) and/or 68 subunits (n=68). In some embodiments, n can range from about 30 to about 60. In some embodiments, n can range from about 35 to about 55. In some embodiments, n can range from about 40 to about 50. In some embodiments, n can range from about 42 to about 48. In some embodiments, n can be 45. In some embodiments, R can be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.
[00282] [00282] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (NOF catalog # GM-020, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG- distearoylglycerol (PEG -DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide and PEG-distearoylglycide, PEG-distearoylglycide, PEG-cholesterol (1-[8'- (Cholest-5-en-3[beta]-oxy)carboxamido-3', 6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega] ]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat. # 880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. # 880120C from Avanti Polar Lipids, Alabaster , Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylenegl icol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA) and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA ). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, disclosed in WO2016/010840 (paragraphs [00240] to [00244]). In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.
[00283] [00283] The LNP may contain (i) an amine lipid for encapsulation and endosomal escape, (ii) a neutral lipid for stabilization, (iii) an auxiliary lipid, also for stabilization, and (iv) a stealth lipid, such as a lipid PEG
[00284] [00284] In some embodiments, an LNP composition may comprise an RNA component that includes one or more of an RNA-guided DNA binding agent, a Cas nuclease mRNA, a Class 2 Cas nuclease mRNA, a Cas9 and a gRNA. In some embodiments, an LNP composition may include a Class 2 Cas nuclease and a gRNA as an RNA component. In certain embodiments, an LNP composition may comprise the RNA component, an amine lipid, a helper lipid, a neutral lipid, and a stealth lipid. In certain LNP compositions, the helper lipid is cholesterol. In other compositions, the neutral lipid is DSPC. In additional embodiments, the stealth lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the LNP composition comprises lipid A or a lipid A equivalent; an auxiliary lipid; a neutral lipid; a stealthy lipid; and a guide RNA. In certain compositions, the amine lipid is lipid A. In certain compositions, the amine lipid is lipid A or an acetal analogue thereof; the helper lipid is cholesterol; the neutral lipid is DSPC; and the stealth lipid is PEG2k-DMG.
[00285] [00285] In certain embodiments, the lipid compositions are described according to the respective molar ratios of the component lipids in the formulation. Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the mol % of the amine lipid can be from about 30 mol % to about 60 mol %. In one embodiment, the mol % of the amine lipid can be from about 40 mol % to about 60 mol %. In one embodiment, the mol % of the amine lipid can be from about 45 mol % to about 60 mol %. In one embodiment, the mol % of the amine lipid can be from about 50 mol % to about 60 mol %. In one embodiment, the mol % of the amine lipid can be from about 55 mol % to about 60 mol %. In one embodiment, the mol % of the amine lipid can be from about 50 mol % to about 55 mol %. In one embodiment, the mol % of the amine lipid may be about 50 mol %. In one embodiment, the mol % of the amine lipid may be about 55 mol %. In some embodiments, the amine lipid in mol % of the LNP batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of the % in target mole. In some embodiments, the mol % amine lipid of the LNP batch will be ± 4 mol %, ± 3 mol %, ± 2 mol %, ± 1.5 mol %, ± 1 mol %, ± 0.5 mol %, or ± 0.25 mol % of the target mol %. All mole % numbers are given as a portion of the lipid component of LNP compositions. In certain embodiments, the LNP batch variability of the amine lipid in mol% will be less than 15%, less than 10%, or less than 5%.
[00286] [00286] In one embodiment, the mol % of the neutral lipid can be between about 5 mol % and about 15 mol %. In one embodiment, the mol % of the neutral lipid can be from about 7 mol % to about 12 mol %. In one embodiment, the mol % of the neutral lipid may be about 9 mol %. In some embodiments, the mol % neutral lipid of the LNP batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, or ± 2.5% of the mol % of target neutral lipid. In certain embodiments, the variability between batches of LNP in moles will be less than 15%, less than 10%, or less than 5%.
[00287] [00287] In one embodiment, the mol % of the auxiliary lipid may be from about 20 mol % to about 60 mol %. In one mode,
[00288] [00288] In one embodiment, the mol % of the PEG lipid can be from about 1 mol % to about 10 mol %. In one embodiment, the mol % of the PEG lipid can be from about 2 mol % to about 10 mol %. In one embodiment, the mol % of the PEG lipid can be from about 2 mol % to about 8 mol %. In one embodiment, the mol % of the PEG lipid can be from about 2 mol % to about 4 mol %. In one embodiment, the mol % of the PEG lipid can be from about 2.5 mol % to about 4 mol %. In one embodiment, the mol % of the PEG lipid may be about 3 mol %. In one embodiment, the mol % of the PEG lipid may be about 2.5 mol %. In some embodiments, the PEG lipid in mol% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the mol% of target PEG lipid. In certain embodiments, the variability between batches of LNP in moles will be less than 15%, less than 10%, or less than 5%.
[00289] [00289] In certain embodiments, the payload includes an mRNA encoding an RNA-guided DNA binding agent (e.g., a Cas nuclease, a Class 2 Cas nuclease, or Cas9) and a gRNA or nucleic acid encoding a gRNA or a combination of mRNA and gRNA. In one embodiment, an LNP composition may comprise a lipid A or its equivalents. In some aspects, the amine lipid is lipid A. In some aspects, the amine lipid is an equivalent of lipid A, for example, an analog of lipid A. In certain aspects, the amine lipid is an acetal analog of lipid A. In various embodiments, an LNP composition comprises an amine lipid, a neutral lipid, an auxiliary lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, the PEG lipid is PEG2k-DMG. In some embodiments, an LNP composition may comprise a lipid A, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, an LNP composition comprises an amine lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from lipid A and an equivalent of lipid A, including a lipid A acetal analog. In additional embodiments, an LNP composition comprises lipid A, cholesterol, DSPC, and PEG2k-DMG.
[00290] [00290] Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the N/P equation. In some embodiments, an LNP composition may comprise a lipid component comprising an amine lipid, an auxiliary lipid, a neutral lipid, and an auxiliary lipid; and a nucleic acid component, wherein the N/P ratio is from about 3 to 10. In some embodiments, an LNP composition may comprise a lipid component comprising an amine lipid, an auxiliary lipid, a neutral lipid, and an auxiliary lipid; and an RNA component, where the N/P ratio is about 3 to 10. In one embodiment, the N/P ratio can be about 5-7. In one embodiment, the N/P ratio can be about 4.5-8. In one embodiment, the N/P ratio can be about 6. In one embodiment, the N/P ratio can be about 6 ± 1. In one embodiment, the N/P ratio can be about 6 ± 0.5. In some embodiments, the N/P ratio will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N/P ratio. In certain embodiments, the variability between batches of LNP in moles will be less than 15%, less than 10%, or less than 5%.
[00291] [00291] In some embodiments, the RNA component may comprise an mRNA, such as an mRNA disclosed herein, for example, encoding a Cas nuclease. In one embodiment, the RNA component may comprise a Cas9 mRNA. In some compositions comprising an mRNA encoding a Cas nuclease, the LNP further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the RNA component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the RNA component comprises a Class 2 Cas nuclease mRNA and a gRNA.
[00292] [00292] In certain embodiments, an LNP composition may comprise an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain LNP compositions comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the neutral lipid is DSPC.
[00293] [00293] In some embodiments, an LNP composition may comprise a gRNA. In certain embodiments, an LNP composition may comprise an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain LNP compositions comprising a gRNA, the helper lipid is cholesterol. In some compositions comprising a gRNA, the neutral lipid is DSPC. In additional embodiments comprising a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalents as an acetal analogue of lipid A.
[00294] [00294] In one embodiment, an LNP composition may comprise an sgRNA. In one embodiment, an LNP composition may comprise a Cas9 sgRNA. In one embodiment, an LNP composition may comprise a Cpf1 sgRNA. In some compositions comprising an sgRNA, the LNP includes an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an sgRNA, the helper lipid is cholesterol. In other compositions comprising an sgRNA, the neutral lipid is DSPC. In additional embodiments comprising an sgRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalents, such as acetal analogues of lipid A.
[00295] [00295] In certain embodiments, an LNP composition comprises an mRNA disclosed herein, which encodes a Cas nuclease, and a gRNA, which may be a sgRNA. In one embodiment, an LNP composition may comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the helper lipid is cholesterol. In some compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease and a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalents, such as acetal analogues of lipid A.
[00296] [00296] In certain embodiments, the LNP compositions include a Cas nuclease mRNA, such as a Class 2 Cas mRNA, and at least one gRNA. In certain embodiments, the LNP composition includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA, of from about 25:1 to about 1:25. In certain embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA, of from about 10:1 to about 1:10. In certain embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA, of from about 8:1 to about 1:8. As measured here, the ratios are by weight. In some embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA such as Class 2 Cas mRNA of from about 5:1 to about 1:5. In some embodiments, the ratio range is from about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments, the gRNA to mRNA ratio is about 3:1 or about 2:1 In some embodiments, the mRNA to gRNA to Cas nuclease ratio, such as Class 2 Cas nuclease, is about 1: 1. The ratio can be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10, or 1:25.
[00297] [00297] The LNP compositions disclosed herein may include a template nucleic acid. The template nucleic acid may be co-formulated with an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid may be co-formulated with a guide RNA. In some embodiments, the template nucleic acid can be co-formulated with an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid may be formulated separately from an mRNA encoding a Cas nuclease or a guide RNA. Template nucleic acid can be delivered with or separately from LNP compositions. In some embodiments, the template nucleic acid may be single-stranded or double-stranded, depending on the desired repair mechanism. The template may have regions of homology to the target DNA or to sequences adjacent to the target DNA.
[00298] [00298] Any of the formulations of LNPs and LNPs described herein are suitable for delivery of an mRNA encoding an RNA-guided DNA binding agent, such as a Cas nuclease, alone or in conjunction with one or more guide RNAs. In some embodiments, an LNP composition is encompassed comprising: an RNA component and a lipid component, wherein the lipid component comprises an amine lipid, a neutral lipid, a helper lipid, and a stealth lipid; and wherein the N/P ratio is about 1-10.
[00299] [00299] In some cases, the lipid component comprises lipid A or its acetal analogue, cholesterol, DSPC and PEG-DMG; and wherein the N/P ratio is about 1-10. In some embodiments, the lipid component comprises: about 40-60% by weight of amine lipid; about 5-15% by weight neutral lipid; and about 1.5 to 10 mol% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In some embodiments, the lipid component comprises: about 50-60% by weight of amine lipid; about 8 to 10% by weight of neutral lipid; and about 2.5-4 mol% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-8. In some cases, the lipid component comprises: about 50-60% by weight of amine lipid; about 5-15 mol% DSPC; and about 2.5-4 mol% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N/P ratio of the LNP composition is about 3-8. In some cases, the lipid component comprises: 48-53% by mole of lipid A; about 8-10 mol% DSPC; and 1.5-10 mol% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N/P ratio of the LNP composition is 3-8 ± 0.2.
[00300] [00300] In some embodiments, LNPs are formed by mixing an aqueous solution of RNA with an organic solvent-based lipid solution, eg 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer, for example for in vivo administration of LNPs, can be used. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at pH 6.5 or above. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at pH 7.0 or above. In certain embodiments, the composition has a pH ranging from about 7.2 to about 7.7. In additional embodiments, the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6. In other embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition can be measured with a micro pH probe.
[00301] [00301] In some embodiments, microfluidic mixing, T-mixing or cross-mixing is used. In certain respects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions and/or RNA and lipid concentrations may vary. LNP or LNP compositions can be concentrated or purified, for example, via dialysis, tangential flow filtration or chromatography. LNPs can be stored as a suspension, emulsion or lyophilized powder, for example. In some embodiments, an LNP composition is stored at 2-8°C, in certain aspects, LNP compositions are stored at room temperature. In additional embodiments, an LNP composition is stored frozen, for example, at -20°C or -80°C. In other embodiments, an LNP composition is stored at a temperature ranging from about 0°C to about -80°C. Frozen LNP compositions may be thawed prior to use, for example on ice, at 4°C, at room temperature or at 25°C. Frozen LNP compositions can be kept at various temperatures, for example on ice, at 4°C, at room temperature, at 25°C or at 37°C.
[00302] [00302] In some embodiments, an LNP composition has greater than about 80% encapsulation. In some embodiments, an LNP composition has a particle size of less than about 120 nm. In some embodiments, an LNP composition has a pdi of less than about 0.2. In some embodiments, at least two of these features are present. In some embodiments, each of these three features is present. Analytical methods for determining these parameters are discussed below in the general section on reagents and methods.
[00303] [00303] In some embodiments, LNPs associated with an mRNA disclosed herein are for use in the preparation of a drug.
[00304] [00304] Electroporation is also a well-known means for charge distribution, and any electroporation methodology can be used for delivery of any of the gRNAs disclosed herein. In some embodiments, electroporation can be used to deliver an mRNA disclosed herein and one or more guide RNAs.
[00305] [00305] In some embodiments, a method is provided for delivering an mRNA disclosed herein to a cell ex vivo, wherein the mRNA is associated with an LNP or not associated with an LNP. In some embodiments, the mRNA/LNP or mRNA is also associated with one or more guide RNAs.
[00306] [00306] In some embodiments, when an mRNA disclosed herein is administered to a mammal in a pharmaceutical composition, the mammal exhibits a cytokine response of at least 1.5, 2, 2.5, 3, 3.5, 4, 4 .5, 5, 5.5, 6, 7, 7.5, 8, 8.5, 9, 9.5 or 10-fold lower than a mammal administered with an mRNA encoding a Cas 9 nuclease with more than 150 % of the minimum uridine content. A cytokine response can be determined as described in the Examples. A difference between cytokine responses can be measured as the mean change in a panel of cytokines such as at least one, two, three or four of the following cytokines: IFN alpha, IL-6, TNF alpha and MCP-1. In some embodiments, when an mRNA disclosed herein is administered to a mammal in a pharmaceutical composition, the mammal exhibits a cytokine response of at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 7.5, 8, 8.5, 9, 9.5 or 10-fold lower than a mammal administered an mRNA with an ORF encoding a Cas 9 nuclease, where the ORF sequence consisting of SEQ ID NO: 5. In some embodiments, uridines in the ORF with a sequence consisting of SEQ ID NO: 5 are not modified. It is generally understood that characteristics of the comparative composition other than mRNA should be held constant, including dose, and that the dose should be in an appropriate range, such as 0.1-5 mpk or other ranges described herein (e.g., as per discussed in the Determining mRNA Efficacy section).
[00307] [00307] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector, transcript, or mRNA comprising the nucleotide sequence encoding the RNA-guided DNA binding agent. In some embodiments, expression of the guide RNA and the RNA-guided DNA binding agent may be driven by their own corresponding promoters. In some embodiments, guide RNA expression may be driven by the same promoter that drives RNA-guided DNA binding agent expression. In some embodiments, the guide RNA and the ORF encoding the RNA-guided DNA binding agent may be contained in a single transcript. For example, the guide RNA may be within an untranslated region (UTR) of the RNA-guided DNA-binding agent transcript. In some embodiments, the guide RNA may be within the 5' UTR of the RNA-guided DNA-binding agent transcript. In other embodiments, the guide RNA may be within the 3' UTR of the RNA-guided DNA-binding agent transcript. In some embodiments, the intracellular half-life of the RNA-guided DNA-binding agent transcript can be shortened by containing the guide RNA within its 3' UTR and thereby shortening the length of its 3' UTR. In additional embodiments, the guide RNA may be within an intron of the RNA-guided DNA-binding agent transcript. In some embodiments, suitable splicing sites may be added in the intron within which the guide RNA is located, so that the guide RNA is properly spliced from the transcript. In some embodiments, expression of the RNA-guided DNA-binding agent and the guide RNA in close proximity to the same vector can facilitate more efficient formation of a RNA-guided DNA-binding agent ribonucleoprotein complex with the guide RNA. .
[00308] [00308] In some embodiments, a pharmaceutical formulation comprising an mRNA is provided in accordance with the disclosure. In some embodiments, a pharmaceutical formulation is provided comprising at least one lipid, for example, an LNP comprising an mRNA in accordance with the disclosure. Any LNP suitable for RNA delivery can be used, such as those described above; examples of additional LNPs are described in PCT/US2017/024973, filed March 30, 3017. A pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier, for example, water or a buffer. A pharmaceutical formulation may further comprise one or more pharmaceutically acceptable excipients, such as a stabilizer, preservative, bulking agent or the like. A pharmaceutical formulation may further comprise one or more pharmaceutically acceptable salts, such as sodium chloride. In some embodiments, the pharmaceutical formulation is formulated for intravenous administration. In some embodiments, the pharmaceutical formulation is formulated for delivery to the hepatic circulation. C. Determination of mRNA efficacy
[00309] [00309] In some embodiments, the effectiveness of an mRNA is determined when expressed along with other components of an RNP, for example at least one gRNA, as a TTR-targeted gRNA.
[00310] [00310] An RNA-guided DNA binding agent with cleavase activity can lead to double-stranded DNA breaks. Non-homologous end-junction (NHEJ) is a process by which double-strand breaks (DSBs) in DNA are repaired by reattaching the break ends, which can produce errors in the form of insertion/delete mutations (indel ). The DNA ends of a DSB are often subjected to enzymatic processing, resulting in the addition or removal of nucleotides on one or both strands before the ends are joined. These additions or removals prior to reunification result in the presence of insertion or deletion (indel) mutations in the DNA sequence at the NHEJ repair site. Many mutations due to indels alter the reading frame or introduce premature stop codons and therefore produce a non-functional protein.
[00311] [00311] In some embodiments, the effectiveness of an mRNA encoding a nuclease is determined based on in vitro models. In some embodiments, the in vitro model is HEK293 cells. In some embodiments, the in vitro model is HUH7 human hepatocarcinoma cells. In some embodiments, the in vitro model is primary hepatocytes, such as human or mouse primary hepatocytes.
[00312] [00312] In some embodiments, the effectiveness of an RNA is measured by the percentage of TTR editing. Exemplary procedures for determining edit percentage are provided in the Examples below. In some embodiments, the TTR edit percentage is compared to the edit percentage obtained when the mRNA comprises an ORF of SEQ ID NO: 5 with unmodified uridine, all other things being equal.
[00313] [00313] In some embodiments, the efficacy of an mRNA is determined using the serum concentration of TTR in a mouse after administration of an LNP comprising the mRNA and a TTR-targeted gRNA, eg SEQ ID NO: 42. In some In these embodiments, the efficacy of an mRNA is determined using the serum concentration of TTR in a mouse after administration of an LNP comprising the mRNA and a TTR-targeted gRNA, e.g. SEQ ID NO: 69. The serum concentration of TTR can be expressed in absolute terms or in % knockdown relative to a sham-treated control. In some embodiments, the effectiveness of an mRNA is determined using percent editing in the liver in a mouse after administration of an LNP comprising the mRNA and a TTR-targeted gRNA, e.g. SEQ ID NO: 42. In some embodiments, an amount effective is capable of achieving at least 50% edit or 50% serum TTR knockdown. Exemplary effective amounts are in the range of 0.1 to 10 mg/kg (mpk), for example, 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk, or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, or 10 mpk.
[00314] [00314] In some embodiments, detection of gene editing events, such as the formation of insertion/deletion ("indel") mutations and homology-directed repair (HDR) events in the target DNA, utilizes linear amplification with a primer labeled and isolating the labeled amplification products (hereinafter referred to as the "LAM-PCR" or "Linear Amplification (LA)" method).
[00315] [00315] In some embodiments, the method comprises isolating cellular DNA from a cell that has been induced to have a double-strand break (DSB) and, optionally, that has been provided with an HDR template to repair the DSB; performing at least one round of linear amplification of the DNA with a labeled primer; isolating the tag-comprising linear amplification products, thereby discarding any amplification product that was amplified with an unlabeled primer; optionally further amplify the isolated products; and analyzing the linear amplification products, or additional amplified products, to determine the presence or absence of an editing event, such as a double-strand break, insertion, deletion, or HDR template sequence in the target DNA . In some cases, the edit event can be quantified. Quantification and the like as used herein (including in the context of HDR and non-HDR edit events such as indels) include detecting the frequency and/or type(s) of edit events in a population.
[00316] [00316] In some embodiments, only one cycle of linear amplification is conducted.
[00317] [00317] In some cases, the labeled primer comprises a molecular barcode. In some embodiments, the labeled primer comprises a molecular barcode and only one round of linear amplification is conducted.
[00318] [00318] In some embodiments, the analysis step comprises sequencing the linear amplified products or additional amplified products. Sequencing may comprise any method known to those skilled in the art, including next generation sequencing and cloning the linear amplification products or additional amplified products into a plasmid and sequencing the plasmid or a portion of the plasmid. In other aspects, the analysis step comprises performing digital PCR (dPCR) or digital droplet PCR (ddPCR) on the linear amplified products or additional amplified products. In other cases, the analysis step comprises contacting the linear amplified products or other amplified products with a nucleic acid probe designed to identify DNA comprising the HDR template sequence and detecting the probes that have bound to the product(s)( s) amplified or additional amplified product(s). In some embodiments, the method further comprises determining the location of the HDR template in the target DNA.
[00319] [00319] In certain embodiments, the method further comprises determining the sequence of an insertion site in the target DNA, wherein the insertion site is the site at which the HDR template is incorporated into the target DNA and wherein the insertion site may include some target DNA sequence and some HDR template sequences.
[00320] [00320] In some embodiments, linear amplification of target DNA with a labeled primer is performed for 1-50 cycles, 1-60 cycles, 1-70 cycles, 1-80 cycles, 1-90 cycles, or 1-100 cycles.
[00321] [00321] In some embodiments, linear amplification of target DNA with a labeled primer comprises a denaturation step to separate DNA duplexes, an annealing step to allow primer binding, and an elongation step. In some embodiments, linear amplification is isothermal (does not require a change in temperature). In some embodiments, isothermal linear amplification is loop-mediated isothermal amplification (LAMP), strand-shift amplification (SDA), helicase-dependent amplification, or enzyme nicking amplification reaction.
[00322] [00322] In some embodiments, the labeled primer anneals to the target DNA at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130 , at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least at least 260, at least 270, at least 280, at least 290, at least 300, at least 1000, at least 5000 or at least 10,000 nucleotides away from the expected editing event site, e.g. insertion, deletion site or model insertion.
[00323] [00323] In some embodiments, the labeled primer comprises a molecular barcode. In some embodiments, the molecular barcode comprises a sequence that is not complementary to the target DNA. In some embodiments, the molecular barcode comprises 6, 8, 10 or 12 nucleotides.
[00324] [00324] In some embodiments, the marker in the primer is biotin, streptavidin, digoxigenin, a DNA sequence, or fluorescein isothiocyanate (FITC).
[00325] [00325] In some embodiments, linear amplification products are isolated using a capture reagent specific to the tag in the primer. In some embodiments, the capture reagent is on a bead, solid support, matrix, or column. In some embodiments, the isolation step comprises contacting the linear amplification product(s) with a tag-specific capture reagent in the primer. In some embodiments, the capture reagent is biotin, streptavidin, digoxigenin, a DNA sequence, or fluorescein isothiocyanate (FITC).
[00326] [00326] In some embodiments, the marker is biotin and the capture reagent is streptavidin. In some embodiments, the label is streptavidin and the capture reagent is biotin. In some embodiments, the tag is at the 5' terminus of the primer, at the 3' terminus of the primer, or internal to the primer. In some embodiments, the tag and/or capture reagent are removed after the isolation step. In some embodiments, the tag and/or capture reagent are not removed and additional amplification and analysis steps are performed in the presence of tag and/or capture.
[00327] [00327] In some embodiments, the additional amplification is non-linear. In some embodiments, the additional amplification is digital PCR, qPCR, or RT-PCR. In some embodiments, the sequencing is next-generation sequencing (NGS).
[00328] [00328] In some embodiments, the target DNA is genomic or mitochondrial. In some embodiments, the target DNA is the genomic DNA of a prokaryotic or eukaryotic cell. In some embodiments, the target DNA is mammalian. The target DNA can be from a cell that does not divide or from a cell that divides. In some embodiments, the target DNA may be from a primary cell. In some embodiments, the target DNA is from a replicating cell.
[00329] [00329] In some cases, cellular DNA is cut before linear amplification. In some embodiments, the cut DNA has an average size of between 0.5 kb and 20 kb. In some cases, cellular DNA is cut to an average length of 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5, 75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.75, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, 12, 0, 12.25, 12.5, 12.75, 13.0, 13.25, 13.5, 13.75, 14.0, 14.25, 14.5, 14.75, 15.05, 15.25, 15.5, 15.5, 15.75, 16.0, 16.25, 16.5, 16.75, 17.0, 17.25, 17.5, 17.75, 18, 0, 18.25, 18.5, 18.75, 19.0, 19.25, 19.5, 19.75 or 20.0 kb. In some cases, cellular DNA is cut to an average length of about 1.5 kb. D. Exemplary uses, methods and treatments
[00330] [00330] In some embodiments, an mRNA, LNP, or pharmaceutical composition is for use in genome editing, for example, in editing a target gene. In some embodiments, an mRNA, LNP, or pharmaceutical composition is for use in modifying a target gene, for example, altering its sequence or epigenetic status. In some embodiments, an mRNA, LNP, or pharmaceutical composition is for use in inducing a double-strand break (DSB) within a target gene. In some embodiments, an mRNA, LNP or pharmaceutical composition is for use in inducing an indel within a target gene. In some embodiments, the use of an mRNA, LNP or pharmaceutical composition disclosed herein is provided for the preparation of a drug for genome editing, for example, editing a target gene. In some embodiments, the use of an mRNA, LNP or pharmaceutical composition disclosed herein is provided for the preparation of a drug to modify a target gene, for example, to alter its epigenetic sequence or status. In some embodiments, the use of an mRNA, LNP, or pharmaceutical composition disclosed herein is provided for the preparation of a medicament for inducing a double-strand break (DSB) within a target gene. In some embodiments, the use of an mRNA, LNP or pharmaceutical composition disclosed herein is provided for the preparation of a medicament for inducing an indel within a target gene. In some embodiments, the target gene is in a subject, such as a mammal, such as a human. In some embodiments, the target gene is in an organ, such as a liver, such as a mammalian liver, such as a human liver. In some embodiments, the target gene is in a liver cell, such as a mammalian liver cell, such as a human liver cell. In some embodiments, the target gene is on a hepatocyte, such as a mammalian hepatocyte, such as a human hepatocyte. In some embodiments, the liver cell or hepatocyte is in situ. In some embodiments, the liver cell or hepatocyte is isolated, for example, in a culture, as in a primary culture. Also provided are methods corresponding to the uses disclosed herein, which comprise administering the mRNA, LNP or pharmaceutical composition disclosed herein to a subject or contacting a cell as described above with the mRNA, LNP or pharmaceutical composition disclosed herein.
[00331] [00331] In some embodiments, an mRNA, LNP, or pharmaceutical composition is for use in therapy or treatment of a disease, e.g., TTR-associated amyloidosis (ATTR). In some embodiments, use of an mRNA disclosed herein (e.g., in a composition provided herein) is provided for the preparation of a medicament, e.g., for the treatment of a subject with TTR-associated amyloidosis (ATTR).
[00332] [00332] In some embodiments, an mRNA, LNP, or pharmaceutical composition is administered intravenously for any of the uses discussed above with respect to organisms, organs, or cells in situ. In some embodiments, an mRNA, LNP, or pharmaceutical composition is administered at a dose in the range of 0.01 to 10 mg/kg (mpk), e.g., 0.01 to 0.1 mpk, 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5 or 10 mpk.
[00333] [00333] In any of the foregoing embodiments involving a subject, the subject may be mammalian. In any of the foregoing embodiments involving a subject, the subject may be human. In any of the foregoing embodiments involving a subject, the subject may be a cow, pig, monkey, sheep, dog, cat, fish, or bird.
[00334] [00334] In some embodiments, an mRNA, LNP or pharmaceutical composition disclosed herein is administered intravenously or for intravenous administration. In some embodiments, the RNAs, compositions and guide formulations are administered into the hepatic circulation or for administration into the hepatic circulation.
[00335] [00335] In some embodiments, a single administration of an mRNA, LNP or pharmaceutical composition disclosed herein is sufficient to disrupt expression of the target gene product. In some embodiments, a single administration of an mRNA, LNP or pharmaceutical composition disclosed herein is sufficient to eliminate expression of the target gene product. In other modalities,
[00336] [00336] In some embodiments, the efficacy of treatment with an mRNA, LNP, or pharmaceutical composition disclosed here is seen at 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years after distribution.
[00337] [00337] In some modalities, treatment slows or halts disease progression.
[00338] [00338] In some modalities, treatment results in improvement, stabilization, or slowing of change in organ function or disease symptoms of an organ, such as the liver.
[00339] [00339] In some modalities, the effectiveness of treatment is measured by the increase in the subject's survival time. E. Exemplary DNA Molecules, Vectors, Expression Constructs, Host Cells, and Production Methods
[00340] [00340] In certain embodiments, the disclosure provides a DNA molecule comprising a sequence encoding any of the mRNAs encoding an RNA-guided DNA binding agent described herein. In some embodiments, in addition to the RNA-guided DNA binding agent sequences, the DNA molecule further comprises nucleic acids that do not encode RNA-guided DNA binding agents. Nucleic acids that do not encode RNA-guided DNA binding agents include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids that encode a guide RNA.
[00341] [00341] In some embodiments, the DNA molecule further comprises a sequence of nucleotides that encodes a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA comprises or consists of a guide sequence flanked by all or part of a repeat sequence of a naturally occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA or crRNA and trRNA. In some embodiments, the crRNA and trRNA are encoded by non-contiguous nucleic acids within a vector. In other embodiments, the crRNA and trRNA may be encoded by a contiguous nucleic acid. In some embodiments, the crRNA and trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and trRNA are encoded by the same strand of a single nucleic acid.
[00342] [00342] In some embodiments, the DNA molecule further comprises a promoter operably linked to the sequence encoding any of the mRNAs encoding an RNA-guided DNA binding agent described herein. In some embodiments, the DNA molecule is an expression construct suitable for expression in a mammalian cell, for example, a human cell or a mouse cell, such as a human hepatocyte or a rodent (e.g., mouse) hepatocyte. In some embodiments, the DNA molecule is an expression construct suitable for expression in a mammalian organ cell, e.g., a human liver or a rodent (e.g., mouse) liver. In some embodiments, the DNA molecule is a plasmid or an episome. In some embodiments, the DNA molecule is contained in a host cell, such as a bacterium or a cultured eukaryotic cell. Exemplary bacteria include proteobacteria such as E. coli. Exemplary cultured eukaryotic cells include primary hepatocytes, including rodent (eg, mouse) or human origin hepatocytes; hepatocyte cell lines, including rodent (eg mouse) or human origin hepatocytes; human cell lines; rodent cell lines (eg, mouse); CHO cells; microbial fungi such as fission or budding yeasts, for example Saccharomyces, such as S. cerevisiae; and insect cells.
[00343] [00343] In some embodiments, a method of producing an mRNA disclosed herein is provided. In some embodiments, this method comprises contacting a DNA molecule described herein with an RNA polymerase under conditions permissive for transcription. In some embodiments, contact is performed in vitro, for example, in a cell-free system. In some embodiments, the RNA polymerase is an RNA polymerase of bacteriophage origin, such as T7 RNA polymerase. In some embodiments, NTPs are provided that include at least one nucleotide modified as discussed above. In some embodiments, the NTPs include at least one nucleotide modified as discussed above and do not comprise UTP.
[00344] [00344] In some embodiments, an mRNA disclosed herein alone, or in conjunction with one or more guide RNAs, may be comprised within or distributed by a vector system of one or more vectors. In some embodiments, one or more of the vectors, or all of the vectors, may be DNA vectors. In some embodiments, one or more of the vectors, or all of the vectors, may be RNA vectors. In some embodiments, one or more of the vectors, or all of the vectors, may be circular. In other embodiments, one or more of the vectors, or all of the vectors, may be linear. In some embodiments, one or more of the vectors, or all of the vectors, may be encased in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Exemplary non-limiting vectors include plasmids, phagemids, cosmids, artificial chromosomes,
[00345] [00345] Exemplary non-limiting viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper-dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors and retrovirus vectors. In some embodiments, the viral vector may be an AAV vector. In other embodiments, the viral vector may be a lentivirus vector. In some embodiments, the lentivirus may not be integral. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high cloning or "unboosted" adenovirus where all viral coding regions, plus the 5' and 3' inverted terminal repeats (ITRs) and the packaging signal ('I' ), are excluded from the virus to increase its packaging capacity. In still other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper-dependent, and in other embodiments, it is helper-independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with structural components for packaging, while a 30kb deleted HSV-1 vector that removes non-essential viral functions does not require helper virus. In additional embodiments, the viral vector may be the T4 bacteriophage. In some embodiments, the T4 bacteriophage may be able to package any linear or circular DNA or RNA molecules when the virus head is emptied. In other embodiments, the viral vector may be a baculovirus vector. In still other embodiments, the viral vector may be a retrovirus vector. In embodiments that use AAV or lentiviral vectors, which have less cloning capability, it may be necessary to use more than one vector to distribute all components of a vector system, as disclosed in this document. For example, an AAV vector may contain sequences that encode a Cas protein, while a second AAV vector may contain one or more guide sequences.
[00346] [00346] In some embodiments, the vector may be capable of directing the expression of one or more coding sequences, such as the coding sequence of an mRNA disclosed herein, in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, for example, a bacterial cell. In some embodiments, the cell can be a eukaryotic cell, such as, for example, a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different cell types are known in the art. In some embodiments, the promoter may be wild-type. In other embodiments, the promoter can be modified for more efficient or effective expression. In still other embodiments, the promoter may be truncated and still retain its function. For example, the promoter can be either a normal size or a reduced size suitable for proper packaging of the vector in a virus.
[00347] [00347] In some embodiments, the vector system may comprise a copy of a nucleotide sequence that encodes an RNA-guided DNA binding agent. In other embodiments, the vector system may comprise more than one copy of a nucleotide sequence that encodes an RNA-guided DNA binding agent. In some embodiments, the nucleotide sequence encoding the RNA-guided DNA binding agent may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one promoter.
[00348] [00348] In some embodiments, the promoter may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Exemplary non-limiting constitutive promoters include cytomegalovirus (CMV) immediate early promoter, simian virus (SV40) promoter, adenovirus major late promoter (MLP), Rous sarcoma virus (RSV) promoter, mammary tumor virus promoter (MMTV), phosphoglycerate kinase (PGK) promoter, alpha elongation factor (EF1a) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any one of the above. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter can be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Exemplary non-limiting inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low level of basal (non-induced) expression, such as the Tet-On® promoter (Clontech).
[00349] [00349] In some embodiments, the promoter may be a tissue-specific promoter, for example, a promoter specific for expression in the liver.
[00350] [00350] The vector may further comprise a nucleotide sequence encoding at least one guide RNA. In some embodiments, the vector comprises a copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be non-identical, so that they target different target sequences, or they may be identical, because they target the same target sequence.
[00351] [00351] In some embodiments, the compositions comprise a vector system, wherein the system comprises more than one vector. In some embodiments, the vector system may comprise a single vector. In other embodiments, the vector system may comprise two vectors. In additional embodiments, the vector system may comprise three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of the guide RNA are used, the vector system may comprise more than three vectors.
[00352] [00352] In some embodiments, the vector system may comprise inducible promoters to initiate expression only after it is delivered to a target cell. Exemplary non-limiting inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics or alcohol. In some embodiments, the inducible promoter may be one that has a low level of basal (non-induced) expression, such as the Tet-On® promoter (Clontech).
[00353] [00353] In additional embodiments, the vector system may comprise tissue-specific promoters to initiate expression only after being delivered to a specific tissue. EXAMPLES
[00354] [00354] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of this disclosure in any way.
[00355] [00355] General Reagents and Methods. Unless otherwise noted, mRNA was synthesized by in vitro transcription (IVT) using a linearized plasmid DNA template and T7 RNA polymerase.
[00356] [00356] For all methods, the concentration of the transcript was determined by measuring the absorbance of light at 260 nm (Nanodrop) and the transcript was analyzed by capillary electrophoresis by Bioanlayzer (Agilent).
[00357] [00357] Unless otherwise indicated, in vivo editing experiments were performed with female CD-1 mice and Charles River Laboratories Sprague-Dawley rats. Unless otherwise indicated, analysis of serum TTR levels in mice was performed as follows. Blood was collected and serum was isolated as indicated.
[00358] [00358] Where indicated in the applicable example, cytokine induction in the treated mice was also measured. For this analysis, approximately 50-100 μL of blood was collected by cutting the tail vein for serum cytokine measurements. The blood was allowed to clot at room temperature for approximately 2 hours and then centrifuged at 1000xg for 10 minutes before collecting serum. A Luminex-based multiplex magnetic bead assay (Affymetrix ProcartaPlus, catalog number Exp040-00000-801) measuring IL-6, TNF-alpha, IFN-alpha and MCP-1 was used for analysis of cytokines collected in the samples. Kit reagents and standards were prepared as indicated in the manufacturer's protocol. Mouse serum was diluted 4-fold using the sample diluent provided and 50 μL was added to wells containing 50 μL of the diluted antibody coated magnetic beads. The plate was incubated for 2 hours at room temperature and then washed. Diluted biotin antibody (50 μL) was added to the beads and incubated for 1 hour at room temperature. The beads were washed again before adding 50 μL of diluted streptavidin-PE to each well, followed by incubation for 30 minutes. The beads were washed once more and then suspended in 100 µL wash buffer and read on the Bio-Plex 200 instrument (Bio-Rad). Data were analyzed using Bioplex Manager ver. analysis package 6.1 with cytokine concentrations calculated from a standard curve using a five-parameter logistic curve fit.
[00359] [00359] Unmodified ATP, GTP, CTP and UTP were used unless otherwise stated. All mRNAs encoded a nuclear localization signal unless otherwise noted.
[00360] [00360] LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblrTM Benchtop Instrument, according to the manufacturer's protocol, or cross-flow mixing as described below. Unless otherwise noted, the LNPs contained 45% lipid A, 9% DSPC, 44% cholesterol and 2% PEG2k-DMG and an N:P ratio of 4.5. LNP Formulation - NanoAssemblr
[00361] [00361] In general, the components of the lipid nanoparticles were dissolved in 100% ethanol with the lipid component of various molar ratios. The RNA loadings were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in an RNA loading concentration of approximately 0.45 mg/mL. LNPs were formulated with an amine lipid to RNA phosphate (N:P) molar ratio of about 4.5 or about 6, with the mRNA to gRNA ratio at 1:1 by weight.
[00362] [00362] The LNPs were formed by microfluidically mixing the lipid and RNA solutions using a Precision Nanosystems NanoAssemblrTM benchtop instrument, according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were collected, diluted in water (approximately 1:1 v/v), kept for 1 hour at room temperature and further diluted with water (approximately 1:1 v/v) before the final buffer exchange. Final buffer exchange in 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) was completed with PD-10 (GE) desalting columns. If necessary, the formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at -80°C until further use. LNP Formulation - Cross Flow
[00363] [00363] For the LNPs prepared using the cross-flow technique, the LNPs were formed by impact jet mixing the lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipid in ethanol is mixed by cross mixing with the two volumes of RNA solution. A fourth stream of water is mixed with the outgoing stream from the cross through an in-line tee. (See WO2016010840 Figure 2.) The LNPs were kept for 1 hour at room temperature and then diluted with water (approximately 1:1 v/v). The diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100kD MWCO) and then buffer exchanged by diafiltration in 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7 .5 (TSS). Alternatively, the final buffer exchange in the TSS was completed with PD-10 (GE) desalting columns. If necessary, the formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4°C or -80°C until further use. Formulation Analysis
[00364] [00364] Dynamic Light Scattering ("DLS") is used to characterize the polydispersity index ("pdi") and size of the LNPs of the present disclosure. DLS measures light scattering resulting from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the particle size distribution (around the average particle size) in a population, with a perfectly uniform population having a PDI of zero. Mean particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted 30X in PBS before being measured by DLS. The mean diameter Z, which is a measure based on the mean particle size intensity, was reported along with the numerical mean diameter and pdi. A Malvern Zetasizer instrument is also used to measure the zeta potential of the LNP. Samples are diluted 1:17 (50 µl to 800 µl) in 0.1X PBS, pH 7.4 prior to measurement.
[00365] [00365] A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is used to determine the total concentration of RNA and free RNA. Encapsulation efficiency is calculated as (total RNA - free RNA)/total RNA. LNP samples are diluted appropriately with 1x TE buffer containing 0.2% Triton-X 100 to determine total RNA or 1x TE buffer to determine free RNA. Standard curves are prepared using the initial RNA solution used to make the formulations and diluted in 1x TE buffer +/- 0.2% Triton-X 100. RiboGreen® dye diluted (according to manufacturer's instructions) is then added to each of the standards and samples and allowed to incubate for approximately 10 minutes at room temperature in the absence of light. A SpectraMax M5 microplate reader (Molecular Devices) is used to read the samples with excitation, auto-cut, and emission wavelengths set to 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA are determined from the appropriate standard curves.
[00366] [00366] Encapsulation efficiency is calculated as (total RNA - free RNA)/total RNA. The same procedure can be used to determine the encapsulation efficiency of a DNA-based filler component. For single-stranded DNA, Oligreen Dye can be used, and for double-stranded DNA, Picogreen Dye.
[00367] [00367] Typically, when preparing LNPs, encapsulation was > 80%, particle size was < 120 nm, and pdi was 0.2. LNP distribution in vivo
[00368] [00368] Unless otherwise noted, female CD-1 mice, aged between 6 and 10 weeks of age, were used in each study. Animals were weighed and grouped according to body weight to prepare dosing solutions based on the average weight of the group. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal (approximately 10 mL per kilogram of body weight). Animals were observed approximately 6 hours post-dose for adverse effects. Body weight was measured 24 hours after administration, and animals were sacrificed at various times by cardiac puncture exsanguination under isoflurane anesthesia. Blood was collected in serum separator tubes or in tubes containing plasma buffered sodium citrate as described herein. For studies involving in vivo editing, liver tissue was collected from the median lobe or three independent lobes (eg, right median lobe, left median, and left lateral) of each animal for DNA extraction and analysis.
[00369] [00369] Mouse cohorts were measured for liver editing by next-generation sequencing (NGS) and serum TTR levels (data not shown). Transthyretin (TTR) ELISA Analysis
[00370] [00370] Blood was collected and serum was isolated as indicated. Total mouse TTR serum levels were determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111). Rat TTR serum levels were measured using a rat specific ELISA kit (Avia Systems Biology catalog code OKIA00159) according to the manufacturer's protocol. Briefly, sera were serially diluted with the kit sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was performed as instructed. NGS sequencing
[00371] [00371] In summary, to quantitatively determine the efficiency of editing at the target site in the genome, genomic DNA was isolated and deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing.
[00372] [00372] The PCR primers were designed around the target site (eg TTR), and the genomic area of interest was amplified. Primer sequences are provided below. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the chemistry needed for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome (eg, hg38) after eliminating those with low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild-type reads versus the number of reads containing an insertion, replacement or deletion was calculated.
[00373] [00373] Edit percentage (e.g. "edit efficiency" or "edit percentage") is defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild type .
[00374] [00374] mRNAs comprising an ORF as set forth in SEQ ID NO: 5 were prepared with a varying content of modified nucleotides, as shown in Table 5 below. The mRNAs were combined with a guide RNA (G282; SEQ ID NO: 42) targeting the transthyretin (TTR) gene and incorporated into LNPs. Unmodified cytidine was used in all LNPs except LNP420. Table 5. LNP417-LNP421 for in vivo studies LNP Cas9 SEQ ID NO Modified nucleotides LNP417 5 N1-methyl-pseudouridine LNP418 5 None
[00375] [00375] LNP417-LNP421 were administered to mice at doses of 0.5 mg/kg (mpk) or 1 mpk. Cytokine induction (IFN alpha, IL-6, TNF alpha and MCP-1) was measured 4 hours post-dose (hpd). The results are shown in Figures 1A-D.
[00376] [00376] At necropsy, 7 days post-dose, serum and liver were collected for measurement of serum TTR and analysis of editing efficacy, respectively. The results are shown in Figures 2A-B.
[00377] [00377] It was observed that the use of pseudouridine and 5-methyl CTP almost completely abolished cytokine induction. The use of 60% (LNP421) or 100% N1-methyl pseudouridine (LNP417) also triggered less cytokine induction than unmodified Cas9 mRNA, and the extent of the 60% reduction of N1-methyl pseudouridine was similar to 100 %.
[00378] [00378] All modified Cas9 constructs were equally effective in lowering serum TTR and were more effective than the unmodified construct, perhaps due to increased stability. According to liver editing data, constructs using pseudouridine and N1-methyl pseudouridine were equally effective. The construct with pseudouridine and 5-methyl cytidine was significantly less effective than pseudouridine alone. The 60% N1-methyl pseudouridine construct may have been slightly less effective than the 100% N1-methyl pseudouridine construct.
[00379] [00379] A Cas9 sequence (SEQ ID NO: 1) was designed to improve liver expression and minimize uridines.
[00380] [00380] In the case of aspartic acid and serine, the hepatic codon corresponding to the highest expression tRNA comprised a thymidine, which would be transcribed as uridine in the corresponding mRNA. The uridine minimal codon was chosen for aspartic acid and serine (GAC and AGC, respectively). The Cas9 ORF sequence was 4140 nt long, contained 528 Us (12.8% uridine content) and avoided running 3 or more consecutive uridines in the ORF. There were 63 cases of UU dinucleotides in the sequence (126/4140 = 3% uridine dinucleotide content). SEQ ID NO: 2 provides an alternative Cas9 sequence that contains 19.6% uridine as an RNA ORF.
[00381] [00381] SEQ ID NO: 3 provides the amino acid sequence of Cas9, which is encoded by SEQ ID Nos: 1 and 2, as the redesign of the Cas9 ORF did not change the encoded amino acid sequence. SEQ ID NO: 4 is the RNA version of the ORF of SEQ ID NO: 1. SEQ ID NO: 5 is the RNA version of the ORF of SEQ ID NO: 2.
[00382] [00382] The effects of modified nucleotides were also evaluated. Modified UTPs used to transcribe the Cas9 transcript included N1-methyl-pseudo-UTP and 5-methoxy-UTP.
[00383] [00383] The structure of N1-methyl-pseudo-UTP is:
[00384] [00384] The structure of 5-methoxy-UTP is:
[00385] [00385] In vitro transcript (IVT) yields were determined for mRNAs comprising ORFs of SEQ ID NO: 4 and
[00386] [00386] These results show that there was a slight decrease in yield as the 5-methoxyuridine content of the mRNA increased, but the mRNA yield was acceptable under all conditions. Thus, Cas9 mRNA can be generated for both Cas9 sequences with acceptable yields under the conditions tested.
[00387] [00387] The purity of the in vitro transcribed mRNAs was calculated using area under the curve (AUC) analysis on capillary electrophoresis (mRNA) tracings of mRNA obtained using an Agilent Bioanalyzer 2100 (Figure 4). The mRNA of SEQ ID NO: 5 Cas9 generated with unmodified UTP generally increased in purity with increasing 5-methoxy-UTP substitution, whereas the same construct made with N1-methyl-pseudo-UTP was less affected by increasing substitution. of 5-methoxy-UTP.
[00388] [00388] SEQ ID NO: 4 Cas9 made with unmodified UTP appeared relatively unaffected by 5-methoxy-UTP substitution, with a slight increase in purity between 0 and 20% substitution with 5-methoxy-UTP.
[00389] [00389] The immunogenicity of different mRNAs was assessed by dot-blot analysis with an anti-dsRNA antibody as a measure of the character of double-stranded (ds) mRNA, an indicator of potential immunogenicity (Figures 5A-D). Figures 5B and 5D used the Cas9 mRNA sequence comprising SEQ ID NO: 5 and Figure 5C used the Cas9 mRNA sequence comprising SEQ ID NO: 4. For constructs generated with unmodified UTP (Figures
[00390] [00390] Editing efficiency was evaluated in vitro by transfecting mRNA together with a guide (G209; SEQ ID NO: 64) targeting transthyretin (TTR) in Neuro 2A cells and measuring percent editing.
[00391] [00391] As shown in Figure 6A, Cas9 mRNA was transcribed from a construct comprising SEQ ID NO: 2 with N1-methyl-pseudo-UTP with 2 nuclear localization sequences and an HA tag (group indicated by bracket leftmost), Cas9 mRNA transcribed from a construct comprising SEQ ID NO: 2 transcribed with UTP with 2 nuclear localization sequences and an HA tag (group indicated by middle), and Cas9 mRNA transcribed from a construct comprising SEQ ID NO: 1 with UTP (group indicated by upper right bracket) were evaluated. For each group, different mRNA concentrations from 0.1 ng-100 ng were evaluated with transcription in increasing amounts of 5-methoxy-UTP from 0% to 100%, as indicated on the X axis. Untreated cells showed no measurable editing. . Figure 6B shows editing efficiency data expressed as EC50 (ng) values.
[00392] [00392] Increasing 5-methoxy-UTP content during transcription appeared to have a negative effect on editing efficiency under the two SEQ ID NO: 5 conditions, with transcripts also containing N1-methyl-pseudo-UTP being more robust than transcripts containing UTP (eg 60% and 80% 5-methoxy-UTP). In contrast, editing efficiency with the Cas9 mRNA sequence comprising SEQ ID NO: 4 showed little or no effect of increasing 5-methoxy-UTP content. Thus, according to this system, the Cas9 mRNA sequence comprising the mRNA of SEQ ID NO: 4 can provide similar editing efficiency with up to 100% 5-methoxyuridine as versions containing unmodified uridine.
[00393] [00393] The in vivo efficacy of the Cas9 mRNA sequence comprising SEQ ID NO: 4 versus the Cas9 mRNA sequence comprising SEQ ID NO: 5 and the transcriptional effect of the Cas9 mRNA sequence comprising SEQ ID NO: 4 in the presence of unmodified UTP, N1-methyl-pseudo-UTP, 40% 5-methoxy-UTP + 60% unmodified UTP or 100% 5-methoxy-UTP were evaluated. Table 7 provides information on these in vivo study groups. Each mRNA was administered as a lipid nanoparticle (LNP) formulation. Table 7. LNP720-LNP724 for in vivo studies LNP Cas9 ORF SEQ ID NO Modified nucleotide LNP720 5 N1-methyl-pseudouridine LNP721 4 N1-methyl-pseudouridine LNP722 4 Unmodified LNP723 4 40% 5-methoxyuridine/60% unmodified LNP724 4 5-methoxyuridine
[00394] [00394] The design of the in vivo study was as follows. CD-1 female mice were from Charles River (n = 5 per group). Animals were dosed intravenously (i.v.) at 1 mg per kilogram (mpk) or 0.5 mpk along with a single guide RNA directed against transthyretin (TTR) (SEQ ID No: 42). Animals that received 1 dose of mpk were bled 4 hours post-dose (hpd) for cytokine analysis of MCP-1, IL-6, IFN-alpha and TNF-alpha. Animals were evaluated at 24 hpd for general welfare. Necropsy was performed 7 days post-dose, with blood collected for serum TTR analysis and liver collected for next-generation sequencing edit analysis.
[00395] [00395] Serum from the 1 mpk dose of animals was collected and 4 hpd, and the serum was prepared and run in the ProcartaPlex® Mouse 4-plex assay (Thermo Fisher) following the manufacturer's instructions. Results for serum levels of MCP-1, IL-6, IFN-alpha and TNF-alpha are shown in Figures 7A-D. These results indicated that the Cas9 mRNA sequence comprising SEQ ID NO: 4 prepared with a modified UTP (LNP721, LNP723 or LNP724) showed relatively low levels of cytokine production.
[00396] [00396] Serum TTR levels were also assessed 7 days post-dose, as shown in Figure 8A and Table 8. The TSS sample (i.e., 5% sucrose, 45 mM NaCl, 50 mM Tris at pH 7,5) indicates TTR levels without LNP treatment. All LNP formulations are described in Table 7. Table 8: Results of serum TTR levels after dosing of LNP720-LNP724 LNP Cas9 ORF SEQ ID NO Modified nucleotide TTR (ug/ml), 0.5 mpk TTR (ug/ml) ), 1 mpk TSS N/AN/A 1019.0 LNP720 5 N1-methyl-pseudouridine 559.4 287.2 LNP721 4 N1-methyl-pseudouridine 160.1 35.3 LNP722 4 Unmodified 483.4 247.0 LNP723 4 40% of 5- 525.8 170.1 methoxyuridine/60% unmodified LNP724 4 5-methoxyuridine 774.0 505.4
[00397] [00397] Table 9 and Figure 8B provide results in terms of percentage of TTR editing in the liver as measured by next generation sequencing (NGS). Table 9: Results as percent edit of liver TTR after LNP720-LNP724 LNP Cas9 dosage SEQ ID NO Modified Nucleotide % Edit, % Edit, 0.5 mpk 1 mpk TSS N/A N1-methyl-pseudouridine 0.16
[00398] [00398] Compared to the TSS control sample, all LNPs comprising Cas9 showed reduction in serum TTR levels and editing above baseline. By comparing the standard Cas9 mRNA (SEQ ID No: 5, LNP720) to the Cas9 mRNA sequence comprising SEQ ID NO: 4 mRNA (SEQ ID No: 4, LNP721), both transcribed with N1-methyl-pseudo-UTP, the Cas9 mRNA sequence comprising SEQ ID NO: 4 showed enhanced activity (lower TTR and higher % edit). For the Cas9 mRNA sequence comprising SEQ ID NO: 4, activity was highest with N1-methyl-pseudo-UTP and transcription with 40% 5-methoxy-UTP + 60% unmodified UTP (LNP723) gave greater activity than with 100% 5-methoxy-UTP (LNP724).
[00399] [00399] As a measure of off-target effects, spleen editing was also measured for animals dosed with 1 mpk of the LNP formulations described above, as shown in Figure 7 and Table 10. For all LNP formulations, either with Cas9 or Cas9 optimised, editing greater than 20 times greater was observed in the liver (Figure 6A). Table 10: Results of spleen TTR percent edit after dosing of 1mpk of LNPs comprising sgRNA and various Cas9 LNP Cas9 SEQ ID NO Modified Nucleotide % Edit, 1 mpk TSS N/AN/A 0.1 LNP720 5 N1-methyl- pseudouridine 0.66 LNP721 4 N1-methyl-pseudouridine 2.42
[00400] [00400] The efficacy of various LNPs was evaluated in vitro in mouse primary hepatocytes (PMHs).
[00401] [00401] At 100 ng, all LNPs described in Table 5 supported TTR editing, as shown in Figure 10. As expected, untreated cells did not show measurable TTR editing.
[00402] [00402] Table 11 shows the EC50 values calculated for each LNP based on the data presented in Figure 10. Table 11: Estimated EC50 values (ng) for TTR gene editing in PMHs LNP EC50 LNP720 45.65 LNP721 23 .04 LNP722 54.00 LNP723 52.40 LNP724 164.1 LNP685 59.88
[00403] [00403] The in vivo efficacy of the Cas9 mRNA sequence comprising SEQ ID NO: 4 versus the Cas9 mRNA sequence comprising SEQ ID NO: 5 was evaluated in mice. Table 12 provides information on these in vivo study groups. Standard Cas9 mRNA refers to SEQ ID No: 5, while mRNA devoid of U (U-dep) refers to SEQ ID No: 4. Each mRNA was administered as a lipid nanoparticle (LNP) formulation.
[00404] [00404] Details of the formulations of LNP716 (Cas9 standard) and LNP738 (devoid of U) are shown in Table 12. Table 12: Characterization of LNP Prep formulation of RNA and RNA Concentration Encapsulation Particle Size LNP ID N:P process ( mg/ml) (%) particle (nm) PDI Citrate-NaCl; X-716 4.5 2.00 98 88.42 0.056 flow_TFF Citrate-NaCl; X-738 4.5 2.22 97 92.80 0.044 flow_TFF PDI = polydispersity index N:P = N:P ratio as described above
[00405] [00405] Serum TTR was measured as described above.
[00406] [00406] Cas9 mRNA with an ORF of SEQ ID NO: 5 was compared to Cas9 mRNA with an ORF of SEQ ID NO: 4 (Figures 11A-B) in mice at doses of 2 mpk and 5 mpk, as shown in Figure 9A and Table 13. These data indicate that ORF Cas9 of SEQ ID NO: 4 induced greater reduction in serum TTR compared to ORF Cas9 of SEQ ID NO: 5 at 2mpk and 5mpk. Figure 9B and Table 13 present these results as percentages relative to the value of the TSS-treated control. The 5 mpk dose of U-dep Cas9 LNP induced a greater than 90% reduction in serum TTR levels. Table 13: Serum TTR levels after administration of LNP716 and LNP738 formulations Cas9 % KD = % knockdown compared to the mean serum concentration of TSS samples.
[00407] [00407] Figure 10 and Table 14 show the liver editing of TTR after administration of LNP716 (standard) and LNP738 (U-dep) formulations at 2mpk and 5mpk. While TSS showed negligible editing, both LNP716 and LNP738 formulations induced hepatic editing of TTR. In comparing the formulations, the LNP738 formulation comprising U depletion induced more than twice the editing of the LNP716 formulation comprising standard Cas9. Table 14: TTR liver editing after dosing with Cas9 formulations devoid of U and standard
[00408] [00408] These data indicate that the U-depleted Cas9 mRNA markedly improved the extent of TTR editing in the liver.
[00409] [00409] The mRNAs encoding Cas9 with UTRs and +/- a hemagglutinin (HA) tag, as indicated in Table 15, were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42)). LNPs were assembled using a Nano AssemblrTM, contained 45% lipid A, 9% DSPC, 44% cholesterol and 2% PEG2k-DMG, were purified using Amicon PD10 filters and used at a concentration of 0.5 mg/ ml (LNP concentration). Female CD-1 mice (n = 5 per group) were dosed i.v. at 0.5 or 1.0 mpk. At 7 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. Table 15. LNP662-LNP669 mRNA descriptions and results of serum TTR and liver edit analyzes Serum TTR Dose (Serum TTR Avg (% LNP mRNA Description Edit (%) (mpk) ug/mL KD mean) TSS - - 944.52 0.06 1 729.56 22.76 20.08 LNP662 ORF:SEQ ID NO: 5; noHA Tag 0.5 988.75 -4.68 8.26 ORF:SEQ ID NO: 45 with tag 1 488.62 48.27 39.12 LNP663 HA 0.5 842.88 10.76 20.18 ORF: SEQ ID NO: 45 with tag 1 628.35 33.47 32.68 LNP664 HA; HBA UTRs 0.5 1087.10 -15.10 14.68 ORF: SEQ ID NO: 45 with tag 1 524.43 44.48 42.70 LNP665 HA; HBB UTRs 0.5 797.37 15.58 18.72 ORF: SEQ ID NO: 45 with tag 1 233.46 75.28 54.28 LNP666 HA; XBG UTRs 0.5 1011.22 -7.06 17.96 ORF: SEQ ID NO: 4; unlabeled 1 197.58 79.08 58.64 LNP667 HA 0.5 689.24 27.03 31.26 ORF: SEQ ID NO: 4; unlabeled 1 622.42 34.10 34.44 LNP668 HA; unmodified NTPs 0.5 811.94 14.04 21.30 ORF: SEQ ID NO: 5; no label 1 1050.68 -11.24 9.82 LNP669 HA; Unmodified NTPs 0.5 1189.70 -25.96 4.04
[00410] [00410] UTRs in mRNAs were HSD/Alb unless otherwise noted. HBA: human alpha globin; HBB: human beta globin (HBB); XBG: xenopus beta globin (XBG). The mRNAs contained 100% N1-methyl pseudouridine in place of uridine, unless otherwise noted.
[00411] [00411] Figures 13A-E show serum TTR (as µg/ml in Figure 13A and % TSS in Figure 13B); liver editing for all LNPs 662-669 (Figure 13C); liver editing for LNP663-LNP666, in which only the UTRs varied (Figure 13D); and liver editing for LNP662 and LNP667-LNP669 in which only the mRNA sequence and UTP modification varied (Figure 13E).
[00412] [00412] Human albumin, human alpha globin, human beta globin and xenopus beta globin RTUs were approximately equally effective; human alpha globin values may be slightly lower, but it was not clear whether the difference was significant.
[00413] [00413] The ORF of SEQ ID NO: 4, which contains less uridines, increased the amount of editing in the liver. Cas9 mRNAs made with N1-methyl pseudouridine were more effective than Cas9 mRNAs made with unmodified uridine.
[00414] [00414] mRNAs comprising an ORF according to SEQ ID NO: 4 or SEQ ID NO: 5 were formulated as LNPs with a TTR-targeted guide RNA, with varying guide weight:Cas9 mRNA ratios, as shown in Table 16. Cas9 mRNA was produced by IVT synthesis, as indicated above, with N1-methylpseudouridine triphosphate instead of uridine triphosphate, HSD 5' UTR, human albumin 3' UTR, and poly-A tail. Table 16. LNPs 815-824 for in vitro and in vivo studies LNP Cas9 SEQ ID NO RNA Ratio (Guide:Cas9) LNP815 5 2:1 LNP816 5 1:1 LNP817 5 1:2 LNP818 5 1:4 LNP819 5 1 :8 LNP820 4 2:1 LNP821 4 1:1 LNP822 4 1:2 LNP823 4 1:4 LNP824 4 1:8
[00415] [00415] Mouse primary hepatocytes (PMH) were seeded in culture medium supplemented with 3% cynomolgus monkey serum for 24 hours and then treated with 0.3, 1, 3 or 10 ng of an LNP shown in Table 16. Cells were lysed after 48 hours and % edit was determined by NGS. The results are shown in Figure 14 and Table 17.
[00416] [00416] For in vivo characterization, LNPs were administered to mice at 0.2, 0.5 or 1 mpk (n = 5 per group). At 8 days post-dose, animals were sacrificed, blood and liver and spleen were collected, and serum TTR, liver edit, and spleen edit were measured. Serum TTR results are shown in Figures 15A-B and Table 18. Liver edit results are shown in Figures 16A-B and Table 19. Spleen edit results are shown in Figures 17A-B and Table 20. Negative control mice were dosed with vehicle (transformation and storage solution; "TSS"). Separate controls were performed for the LNP815-LNP819 experiments and for the LNP820-LNP824 experiments. Table 18. Serum TTR levels after administration of LNP815- LNP824 LNP Ratio Dose (mpk) Serum TTR (ug/mL) Serum TTR (%KD) Guide:Cas9 TSS 974.23 - 1 300.32 69.17 LNP815 2:1 0.5 539.37 44.64 0.2 800.85 17.80 1 183.61 81.15 LNP816 1:1 0.5 466.63 52.10 0.2 859.05 11.82 1 117.86 87.90 LNP817 1:2 0.5 487.26 49.99
[00417] [00417] LNP820-LNP824 generally gave liver editing results greater than or approximately equal to their LNP815-LNP819 counterparts with the same ratio. The LNP820-LNP824 showed consistent performance across the ratio range tested at 0.5 and 1 mpk and across ratios from 2:1 to 1:4 at 0.2 mpk. Table 20. Spleen edit after LNP815-824 LNP administration Guide Ratio:Cas9 Dose (MPK) % TSS Edit 0.12 1 0.6 LNP815 2:1 0.5 0.62 0.2 0.28 1 0.74 LNP816 1:1 0.5 1 0.2 0.28 1 0.74 LNP817 1:2 0.5 0.58 0.2 0.22 1 1.22 LNP818 1:4 0.5 0, 44 0.2 0.3 1 0.9 LNP819 1:8 0.5 0.64 0.2 0.36 TSS 0.225 1 0.83 LNP820 2:1 0.5 0.825 0.2 0.525 1 1.425 LNP821 1: 1 0.5 0.9 0.2 0.425 1 1.85 LNP822 1:2 0.5 0.625 0.2 1.74
[00418] [00418] Additional groups of mice (n = 2) were dosed at 3 mpk with each formulation and sacrificed at 6 hpd for determination of liver protein expression. A Western blot of liver protein from mice treated with 3 mpk from 1:1 and 1:4 ratio formulations (LNP816, LNP818, LNP821 and LNP823) is shown in Figure
[00419] [00419] The mRNAs encoding Cas9 and containing modified nucleotides, as indicated in Table 21, were formulated as LNPs with a TTR-directed guide RNA (G282; SEQ ID NO: 42). LNP1034 contained a Cas9 mRNA commercially obtained from Trilink Biotechnologies, LLC and included a CleanCapTM (Cap1 structure in which the first nucleotide after 7-methylguanine capping is 2'-O-methylated). LNP1027-LNP1033 contained an mRNA comprising an ORF according to SEQ ID NO: 4 and an ARCA (anti-reverse capping analog) Cap0. LNPs were assembled using a Nano AssemblrTM, contained 45% lipid A, 9% DSPC, 44% cholesterol and 2% PEG2k-DMG, purified using Amicon PD10 filters and suspended in TSS buffer. The N:P (nitrogen to phosphate) ratio in the LNPs was 4.5 and the RNA concentration of the formulations was 0.4 mg/ml. Female CD-1 mice (n = 5 per group) were dosed i.v. at 0.1 or 0.3 mpk. At 7 days after the dose, the animals were sacrificed, blood and liver were collected and the
[00420] [00420] For LNPs in which the modified uridine and/or cytidine nucleotides are listed at 25% or 50%, the remaining uridine and/or cytidine nucleotides, respectively, have not been modified.
[00421] [00421] Serum TTR results are shown in Figure 19A-B (serum TTR results expressed in µg/mL and % TSS control, respectively); Figure 20 (liver edit); and Table 22. Table 22. Serum TTR and Liver Edit Results for LNPs 1027-1034 Dose Nucleotide(s) Serum TTr Serum TTR (% % Mean of
[00422] [00422] LNP1027 N1-methyl pseudouridine-containing mRNA had slightly higher editing efficiency compared to LNP1032 pseudouridine-containing mRNA. The potency of mRNA containing pseudouridine and 5-methylcytidine (LNP1033) was greatly reduced. The mRNA containing 25% of 5-iodouridine showed editing efficiency equivalent to the mRNA containing N1-methyl pseudouridine. With 5% 50% iodouridine, there was a reduction in potency. Trilink 5-methoxyuridine mRNA showed low activity.
[00423] [00423] This study evaluated the in vivo efficacy in mice of ARCA-capped Cas9 mRNAs with HBB (human beta-globin) 5' and 3' UTRs; 5' and 3' XBG (xenopus beta-globin) UTRs; or with the 5' human HSD17B4 UTR (HSD) and the albumin 3' UTR (ALB).
[00424] [00424] Formulations containing guide RNA targeting the mouse TTR gene (G534; SEQ ID NO: 72) and Cas9 mRNA in a 1:1 molar ratio in LNPs were prepared using the cross-flow process described above and membrane filtered. VivaFlowTM 50. The LNPs contained cationic lipids (lipid A), cholesterol, DSPC and PEG2k-DMG in a molar ratio of 45:9:43:3 and had an N:P ratio of 6.0.
[00425] [00425] Liver editing and serum TTR results are shown in Figure 21A-C and Table 23. Table 23. Liver editing and serum TTR results in mice with LNP1058-LNP1060. Serum TTR Edition Serum TTR (% LNP UTRs Dose (mpk) liver (%) (µg/ml) KD) TSS 0.0 1366.9 1 66.3 84.4 93.8 1058 HBB (3' and 5' ) 0.3 27.6 881.1 35.5 1 69.1 63.0 95.4 1059 XBG (3' and 5') 0.3 31.6 748.7 45.2 1 62.6 115, 6 91.5 1060 HSD (5') and ALB (3') 0.3 20.9 896.0 34.4
[00426] [00426] Results indicate that all mRNAs tested in LNP1058-LNP1060 were able to support editing. The highest level of editing and the greatest reduction in serum TTR were observed with the mRNA containing XBG UTRs in LNP1059.
[00427] [00427] This study evaluated the in vivo efficacy in mice of different gRNA to mRNA ratios. CleanCap™ capped Cas9 mRNAs with the ORF of SEQ ID NO: 4, HSD 5' UTR, human albumin 3' UTR, a Kozak sequence, and a poly-A tail were produced by IVT synthesis as indicated in
[00428] [00428] LNP formulations were prepared from the described mRNA and sg282 (SEQ ID NO: 42; G282) as described in Example 2 with lipid A, cholesterol, DSPC and PEG2k-DMG in the molar ratio of 55:33:9 :3 and with an N:P ratio of 6. The weight ratios of gRNA:Cas9 mRNA of the formulations were shown in Table 24. Table 24. Characterization of LNP1110-LNP1116. RNA Conc Numerical Mean PDI Size LNP ID EE (%) (mg/mL) particle (nm) particle (nm) 1110 0.92 99 69.52 0.022 56.47 1111 0.86 97 76.65 0.065 57 .36 1112 0.90 99 76.58 0.036 63.11 1113 0.97 99 76.60 0.071 58.92 1114 1.05 99 76.34 0.018 62.82 1115 0.65 99 82.64 0.018 66. 1116 0.75 100 82.01 0.039 65.05
[00429] [00429] For in vivo characterization, the above LNPs were administered to mice with 0.1 mg total RNA (mg lead RNA + mg mRNA) per kg (n = 5 per group). At 7-9 days post-dose. Animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured as described above. Serum TTR and liver edit results are shown in Figure 22A and Figure 22B. Negative control mice were dosed with TSS vehicle.
[00430] [00430] In addition, the above LNPs were administered to mice with a constant mRNA dose of 0.05 mg mRNA per kg (n = 5 per group), while varying the gRNA dose from 0.06 mg per kg to 0.4 mg per kg. At 7-9 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. Serum TTR and liver edit results are shown in Figure 22C and Figure 22D. Negative control mice were dosed with TSS vehicle.
[00431] [00431] Cas9 sequences using different codon schemes were designed to test for improved protein expression. Each sequence was designed to encode the Cas9 amino acid of SEQ ID No: 3 using a distinct set of codons. In each open reading frame sequence, a single codon was used to encode each amino acid. Sequences vary based on how often codons occur in complete protein-coding genes in Homo sapiens based on NCBI-GenBank Flat File Release 160.0 (Nakamura et al. (2000) Nucl. Acids Res. 28, 292; Benson et al. (2006) Nucleic Acids Res. 34(Database issue), D16-20) and the abundance of a particular nucleotide between the codons. Based on the codon schemes shown in Table 4, seven different open reading frames for Cas9 (SEQ ID NO: 52, 54 and 108-112) were constructed that encode the Cas9 protein of SEQ ID NO: 3. They were incorporated into constructs also containing the HSD 5' UTR (SEQ ID NO: 41), an albumin 3' UTR, a T7 promoter and a polyA tail. An exemplary sequence containing the 3'UTR and polyA tail is SEQ ID NO:53, in which the 3'UTR and polyA tail follows the 5'UTR HSD and ORF of SEQ ID NO:52. Also included in these evaluations was included a similarly composed construct using a codon scheme based on optimal codons to improve mRNA half-life, as described by Presnyak and colleagues (2015) (SEQ ID No: 107, using the long half-life codon set of Table 4) to encode the Cas9 protein of SEQ ID NO: 3.
[00432] [00432] Messenger RNA was produced for each construct by IVT using 100% N1-methyl pseudouridine instead of uridine. HepG2 cells were transfected with 800 ng of each Cas9 mRNA using Lipofectamine™ MessengerMAX™ Transfection Reagent (ThermoFisher). Six hours after transfection, cells were lysed by freeze-thaw and discarded by centrifugation. Cas9 protein levels were determined by the ELISA assay. Briefly, the total protein concentration was determined by the bicinchoninic acid assay. A 96-well GOLD Streptavidin SECTOR MSD plate (Meso Scale Diagnostics, Cat. L15SA-1) was prepared according to the manufacturer's protocol, using Cas9 antibody (Origene, Cat. CF811179) as capture antibody and Cas9 mAb (7A9 -3A3). (Cell Signaling Technology, Cat. 14697) as the detection antibody. Recombinant Cas9 protein was used as a calibration standard in Diluent 39 (Meso Scale Diagnostics) with 1X Halt™ Protease Inhibitor Cocktail, EDTA-Free (ThermoFisher, Cat. 78437). ELISA plates were read using Meso Quickplex SQ120 instrument (Meso Scale Discovery) and data were analyzed with Discovery Workbench 4.0 software package (Meso Scale Discovery).
[00433] [00433] Editing efficiency was evaluated in vitro by transfecting mRNA in conjunction with a guide (G502; SEQ ID NO: 70) targeting transthyretin (TTR) in HepG2 cells and measuring percent editing. Cas9 mRNAs comprising SEQ ID Nos indicated in Table 25 were evaluated at mRNA concentrations of 3 ng-100 ng. Untreated cells showed no measurable editing. Figures 23-24 and Table 25 show the effects of different sets of codons on Cas9 protein expression and editing in vitro. Table 25. In vitro editing and expression of ORFs with different sets of codons. ng Cas9/mg ng Cas9/mg % Edit (30 ng Deviation ORF (codon set) protein total protein standard mRNA of total standard deviation transfected) edit SEQ ID No: 50 (Table 6 codons 10.99 1.04 35.6 2.11 ng Cas9/mg ng Cas9/mg % Edit (30 ng Deviation ORF (codon set) protein total protein standard mRNA of total standard deviation transfected) min uridine edit, splice junctions removed) SEQ ID No: 107 18 .78 2.83 36.5 3.27 (Table 4 long half-life) SEQ ID No: 52 (Table 4 U1 low) 31.23 4.47 22.2 2.83 SEQ ID No: 54 (Table 4 U2 low) 1.54 0.16 14.7 0.40 SEQ ID No: 108 (Table 4 U high) 1.41 0.12 14.0 2.95 SEQ ID No: 109 (Table 4 G low) 4 .95 0.70 19.6 2.29 SEQ ID NO: 110 (Table 4 C below) 2.26 0.16 23.1 4.07 SEQ ID No: 111 (Table 4 A below) 74.62 15, 53 41.3 3.56 SEQ ID NO: 112 77.32 10.60 34.8 7.32 (Table 4 A/U low) SEQ ID NO: 4 (Table 6 codons 17.16 1.54 34.7 1.15 uridine minima)
[00434] [00434] To determine the effectiveness of codon schemes in vivo, the expression of the Cas9 protein was measured when expressed in vivo from mRNAs encoding Cas9 using codon schemes described in Table 4. Messenger RNAs as indicated in Table 26 , were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42). LNPs were assembled using the cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had an N:P ratio of 6.0. LNPs were purified using Amicon PD-10 filters (GE Healthcare) and used at a concentration of 0.32 mg/ml (LNP concentration). Female CD-1 mice (n = 5 per group) were dosed i.v. at 1 mpk. 3 hours after the dose, the animals were sacrificed, the liver was harvested and the Cas9 expression in the liver was measured. Cas9 protein expression was measured in the liver using the Meso Scale Discovery ELISA assay described above. Approximately 40-50 mg of liver tissue was bead milled in RIPA buffer (Boston Bioproducts BP-115) with 1x complete protease inhibitor tablet (Roche, Cat.11836170001). Figure 25 and TABLE 26 show the results of Cas9 expression in the liver. The mRNAs for the low A and low A/U codon schemes (ORFs from SEQ ID NOs: 111 and 112) showed the highest Cas9 expression of the ORFs tested. The expression of the negative control Cas9 protein and the ORF of SEQ ID NO: 54 were below the lower limit of quantification (LLOQ). Table 26 Mean Cas9 ORF (ng/g liver) Standard Deviation TSS < LLOQ 0.0 SEQ ID NO: 4 1644 1172 SEQ ID NO: 52 1562 951 SEQ ID NO: 54 < LLOQ 0.0 SEQ ID NO: 111 2630 730 SEQ ID NO: 112 2134 362
[00435] [00435] To determine the effectiveness of codon schemes in vivo, genome editing was measured in vivo from mRNAs encoding Cas9 using different codon schemes. Messenger RNAs, as indicated in Table 27, were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42). LNPs were assembled using the cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had a N:P ratio of 6.0. LNPs were purified using Amicon PD-10 filters (GE Healthcare) and used at a concentration of 0.05 mg/ml (LNP concentration). Female CD-1 mice (n = 5 per group, except n = 4 for the group treated with SEQ ID NO: 52) were dosed i.v. at 0.1 mpk. At 6 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. TABLE 27 and Figure 26 show the results of in vivo editing. TABLE 27 and Figures 27A-B show serum levels of TTR. Table 27 Deviation % Mean Serum TTR Standard Deviation ORF Standard Edition n (µg/ml) of Serum TTR TSS Edition 0.06 0.05 856 68 5 SEQ ID No: 4 40.96 8.41 329 143 5 SEQ ID No: 107 44.28 11.45 255 97 5 SEQ ID No: 52 60.10 8.07 143 78 4 SEQ ID No: 54 1.50 0.66 822 161 5 SEQ ID No: 108 0.74 0, 36 914 182 5 SEQ ID No: 111 57.26 4.15 216 62 5 SEQ ID No: 112 61.44 4.50 100 79 5
[00436] [00436] To determine the effectiveness of codon schemes at different mRNA concentrations, an in vivo dose-response experiment was performed. Messenger RNAs, as indicated in Table 28, were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42). LNPs were assembled using the cross-flow method and contained 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG. LNPs were purified using Amicon PD-10 filters (GE Healthcare and used at a concentration of 0.7 mg/ml (LNP concentration). Female CD-1 mice (n = 5 per group) were administered iv at 0, 03, 0.1 or 0.3 mpk. At 7 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. TABLE 28 and Figure 28 show the results of in vivo editing TABLE 28 and Figures 29A-B show serum levels of TTR.
[00437] [00437] To determine the effectiveness of codon schemes with different UTRs, genome editing was measured in vivo after administration of mRNAs encoding Cas9. Messenger RNAs, as indicated in Table 29, were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42). LNPs were assembled using the cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had an N:P ratio of 6.0. LNPs were purified using Amicon PD-10 filters (GE Healthcare) and used at a concentration of 0.05 mg/ml (LNP concentration). Female CD-1 mice (n = 5 per group; n = 4 for editing SEQ ID No: 43) were administered i.v. at 0.1 mpk. At 6 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. TABLE 29 and Figures 30A-B show the results of in vivo editing (B) and serum TTR (A).
[00438] [00438] The mRNAs encoding Cas9 and containing caps, UTRs, and polyA tails, as indicated in Table 30, were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42). LNPs were assembled using the cross-flow procedure, containing 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had N:P ratio 6.0. LNPs were purified using Amicon PD-10 filters (GE Healthcare) and used at a concentration of 0.06 mg/ml (LNP concentration). Female CD-1 mice (n = 5 per group) were dosed i.v. at 0.1 or 0.3 mpk. At 7 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. Figure 31 and Table 30 show that Cap 1 mRNAs have ~10% higher average editing than Cap 0 mRNAs at the 0.1 mpk dose. At the dose of 0.3 mpk, mRNAs with XBG UTR have a slightly higher average edit than mRNA with HSD UTR, with the exception of enzyme cap 0. Serum TTR results are shown in Figure 32 (serum TTR results expressed in µg/mL and the % of TSS control, respectively); Figure 31 (liver edit); and Table 30.
[00439] [00439] Cas9 sequences using various nuclear localization signals (NLSs) were designed and tested to determine effectiveness. Eleven non-canonical NLSs of varying strengths were chosen from those identified by Kosugi et al. (2009) Journal of Biological Chemistry, 284(1), 478–485, as shown in Table 31. These amino acid sequences were added to the carboxy terminus of the Cas9 amino acid sequence (SEQ ID No: 13). The control sequence encodes SEQ ID No. 4.
[00440] [00440] The mRNAs encoding Cas9 with NLSs, as indicated in Table 31, were formulated as LNPs with a TTR-targeted guide RNA (G282; SEQ ID NO: 42). LNPs were assembled using the cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had a N:P ratio of 6.0. LNPs were purified using Amicon PD-10 filters (GE Healthcare) and used at a concentration of about 0.07 mg/ml (LNP concentration). Female CD-1 mice (n = 5 per group) were dosed i.v. at 0.1 mpk. At 7 days post-dose, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured. The results are shown in Table 32 and in Figure
[00441] [00441] The NLS5 showed a statistically significant increase over the SV40 NLS (One-Way ANOVA, p=0.006). NLS4 and NLS8 exhibited a possible trend of increased editing compared to the SV40 NLS, but the difference in this experiment was not statistically significant. Figures 34A-B show serum levels of TTR after administration of nuclear localization signal variants. Kosugi et al. (2009), supra, rate activity of NLSs (“Strength of NLS” in Table 32) by degree of nuclear localization, with 10 as exclusively nuclear and 1 as diffuse throughout the cell. NLS activity rated in this document is positively correlated with editing efficiency, as shown in Figure 35.
[00442] [00442] Table 33 and Figure 36 show the expression of Cas9 from transcripts with different 5' UTRs. All constructs used human albumin 3' UTR. Messenger RNA was produced for each construct by IVT. Messenger RNA for SEQ ID No: 179 was produced using linearized plasmid and all others were generated using the PCR product as a template. HepG2 cells were transfected with 100 ng of each Cas9 mRNA and guide (G502; SEQ ID NO: 70) targeted to the final concentration of 25nM transthyretin (TTR) using Lipofectamine™ MessengerMAX™ Transfection Reagent (ThermoFisher). Six hours after transfection, cells were lysed by the Nano-Glo® HiBiT Lytic assay (Promega). Cas9 protein levels were determined using the Nano-Glo® Nano-Glo HiBiT Extracellular Detection System (Promega, Cat. N2420). Table 33 and Figure 36 show Cas9 expression of transcripts with different 5' UTRs. Table 33: Expression of Cas9 5' UTR mRNA construct Cas9 molecules (107) Standard Deviation (107)
[00443] [00443] Three studies were conducted with LNP formulations prepared as described above, using the X-flow/TFF process. Specific mole loads and amounts are given in Tables 34-36. Each formulation containing Cas9 mRNA and guide RNA (gRNA) had an mRNA:gRNA ratio of 1:1 by weight. LNP doses (in mg/kg, total RNA content), route of administration and whether the animals received pretreatment with dexamethasone are indicated in the Tables. For animals that received pretreatment with dexamethasone (Dex), Dex was administered at 2 mg/kg by intravenous bolus injection 1 hour before LNP or vehicle administration.
[00444] [00444] For chemical analysis of blood, blood was collected from animals at times as indicated in the Tables for each factor measured. Cytokine induction was measured in pre- and post-treatment NHPs. A minimum of 0.5 mL of whole blood was collected from a peripheral vein of conscious and restricted animals in a 4 mL serum separator tube. The blood was allowed to clot for a minimum of 30 minutes at room temperature, followed by centrifugation at 2000xg for 15 minutes. Serum was aliquoted into 2 polypropylene microtubes of 120 uL each and stored at -60 to -86°C until analysis. A custom Meso Scale Discovery (MSD) non-human primate U-Plex Cytokine kit was used for analysis. The following parameters were included in the analysis: INF-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, MCP-1 and TNF-a, with a focus in IL-6 and MCP-
[00445] [00445] Complement levels were measured in pre- and post-treatment animals by enzyme immunoassay. A volume of 0.5 mL of whole blood was collected from a peripheral vein of conscious and restricted animals in a 0.5 mL tube of k2EDTA. The blood was centrifuged at 2000xg for 15 minutes. Plasma was aliquoted into 2 polypropylene microtubes of 120 uL each and stored at -60 to -86°C until analysis. A Quidel MicroVue Complement Plus EIA kit (C3a-Cat # A031) or (Bb-Cat # A027) was used for analysis. Kit reagents and standards were prepared as indicated in the manufacturer's protocol. The plates were run on an MSD Sector Imager 6000 with optical density at 450 nm. Results were analyzed using a 4-parameter curve fit.
[00446] [00446] Data for cytokine induction and complement activation are provided in the tables below. "BLQ" means below the limit of quantification. Lead RNA SEQ ID NOs are as follows: G502, SEQ ID NO: 70; G506, SEQ ID NO: 197; G509, SEQ ID NO: 71; G510, SEQ ID NO: 198. Table 34: Study 1 Molar Ratios (Lipid A, Size, Dose Level, Cholesterol Group, of N:P Load Via Total Dex Content DSPC Treatment and Sample RNA (mg/kg) PEG2k- DMG, (n) respectively (1) TSS IV - n/an/an/a 3 n/a no (vehicle) Cas9 mRNA infusion (2) (SEQ ID IV - LNP699 45/44/9/2 4.5 3 3 no NO:48); G502 infusion G000502 Cas9 mRNA (3) (SEQ ID IV - LNP688 45/44/9/2 4.5 3 3 no NO:48); G506 infusion G000506 Cas9 mRNA (4) (SEQ ID IV - LNP689 45/44/9/2 4.5 3 3 no NO:48); G509 infusion G000509 Cas9 mRNA (5) (SEQ ID IV - LNP690 45/44/9/2 4.5 3 3 no O: 48); infusion G510 G000510 Table 35. Study 2 Molar Ratios Size Level Group of (lipid A, dose, treatment content- cholesterol, DSPC N:P Load Total via dex sample ment and PEG2k-DMG, RNA (n) respectively (mg/kg) (1) TSS IV- n/an/a 1 n/a yes (vehicle) bolus (2) TSS IV- n/an/a 1 n/a no (vehicle) bolus (3) 45 /44/9/2 4.5 Cas9 mRNA 1 IV - 3 yes
[00447] [00447] Cas9 expression was measured in vivo after administration of different mRNAs encoding Cas9. Messenger RNAs, as indicated in Table 49, were formulated as LNPs with a mouse sgRNA targeting the mouse TTR gene (sgRNA:mRNA weight ratio 1:2). LNPs were assembled using the cross flow procedure with 50% lipid A, 9% DSPC, 38% cholesterol and 3% PEG2k-DMG and an N:P ratio of 6.0. LNPs were purified using Sartocon Slice 200 (Sartorius) and used at a concentration of 1.53 mg/ml (RNA concentration). LNP formulations were analyzed for mean particle size, polydispersity (pdi), total RNA content and RNA encapsulation efficiency as described above (data not shown).
[00448] [00448] Female CD-1 mice (n = 5 per group) were dosed i.v. at 0.3 mpk. After 1 hour, 3 hours and 6 hours post-dose, animals were sacrificed, liver tissue was harvested and Cas9 protein levels were measured by MSD ELISA as described in Example 11. Table 49 shows Cas9 protein levels. At each time point, more Cas9 protein is detected in animals treated with SEQ ID NO:177 than in animals treated with SEQ ID NO:43. Table 49 mRNA Time Point ng Cas9/g Deviation Size of (hours) Standard Liver Sample (n) TSS 0 28 5 SEQ ID NO: 43 1 429 164 5 SEQ ID NO: 177 1 1872 907 5 SEQ ID NO: 43 3 1167 814 5 SEQ ID NO: 177 3 2233 929 5 SEQ ID NO: 43 6 535 297 5 SEQ ID NO: 177 6 1663 443 5 Different mRNA dose response comparison
[00449] [00449] The dose response curves of different mRNAs encoding Cas9 in vivo were compared. LNP formulations were prepared with the mRNAs of SEQ ID NO:43 and SEQ ID NO:177 and sg502 (SEQ ID NO:70; G502), formulating as described in Example
[00450] [00450] For in vivo characterization, female CD-1 mice (n = 5 per group) were dosed intravenously at 0.03, 0.1 or 0.3 mg of total RNA (mg lead RNA + mg mRNA ) per kg (n=5 per group). Seven days after dosing, animals were sacrificed, blood and liver were collected, and serum TTR and liver edit were measured as described in Example 1. Negative control animals were dosed with TSS vehicle. Editing data is provided in Table 50, below. For SEQ ID NO: 43, the average of 8 in vivo experiments, each with 5 animals, is provided. For SEQ ID NO: 177, the average of an in vivo experiment with 5 animals at each dose is provided. At each dose, the % edit is greater in animals treated with SEQ ID NO: 177 than in animals treated with SEQ ID NO: 43. Table 50 % Edit 0.3 mg/kg dose 0.1 mg/kg 0, 03 mg/kg mRNA Mean Mean Mean (Range) (Range) (Range) SEQ ID NO: 43 65.8% 40.6% 11.4% (62.2-71.2%) (29.2-55 .6%) (6.2-20.1%) SEQ ID NO: 177 71.2% 58.9% 29.3% Sequence table
[00451] [00451] The following sequence table provides a list of the sequences disclosed herein. It is understood that if a DNA sequence (comprising Ts) is referenced in relation to an RNA, Ts must be replaced by Us (which may or may not be modified depending on the context) and vice versa. Description Sequence SEQ ID No. Sequence of ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGG 1 encoding 2 ATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGT Cas9 DNA CCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCAC TGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACA GCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAA ATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTG GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGAT CTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAAT CTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGA GACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCC TGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTC
Description Sequence SEQ ID No. ATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAAC GCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAG CAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACG GACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCA AGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGAC ACATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTA CGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGA GCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCA AGCATGATCAAAGATACGACGAACACCACCAGGACCTGACACTGCTGAAG GCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGAC CAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGA AGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGA AGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAA CATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCAC GCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGA GAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCG CTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGA AACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACG AAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACA ACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAA ACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCG AATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCC TGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGA CACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCAC ACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAG GATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAG AGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAAACAG AAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACAT CCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTC AAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAA CATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGG GAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAAC
Description Sequence SEQ ID No. GAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGAC CAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTC GTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATC ACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAG CGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGAC AGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGT ACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGA GCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAG AAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCG GAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACG GAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAG GAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACT TCTTCAAGACAGAAATCACACTGGCAAACGGAGAAAATCAGAAAGAGACCGC TGATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGA AGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTC GTCGCAAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGA ACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGAT CGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCAT CAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAAGGAAGAAAGAGAAT GCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGA GCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGG GAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAG CACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTC ATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCA CAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCAC ACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAAT CGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGAT CCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCT
GGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAG Sequence of ATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGT 2 encoding 1 TGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTC Cas9 DNA CTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTG
Description Sequence SEQ ID No. CTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGC GAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGA TCTTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGG AAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCT TTGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCT ACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGAT TGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGAT CGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCA ACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAG CGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGG CGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACT TTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCC AATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTAC GACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGAT CTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATC CTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATT AAGCGGTACGACGAGCATCACCAGGATTCCACGCTGCTCAAAGCGCTCGT GAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAA GAATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCT ATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGC TGGTCAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACA ACGGATCCATTCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGC GGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATC GAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCG CGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCAC TCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTT CATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCT TCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACT AAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGA GAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTG ACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGAC TCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAAC CTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGA GGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGA GGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGA CGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCC GCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAA
Description Sequence SEQ ID No. CTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCA ATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACA AGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCG GTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGAC GAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGA AATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCG AAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATC CTGAAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTA CCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGA CATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTT CTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAAC AGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAA GAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTT TGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGG CTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCAC GTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGAT AAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCG GACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACC ATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCA AAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTA CGACGTGAGGAAGATGATAGCCAAGTCCCGAACAGGAAATCGGGAAAGCAA CTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATT ACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGG AGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTC GCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAACCGAAGTGC AAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACA AGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTC GATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAA GGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCAT CATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAA GGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTACTC ACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAG AACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCC TCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACG AACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCA TCGAACAAATCTCCGAGTTTTCAAAAGCGCGTGATCCTCGCCGACGCCAACC TCCAAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAG
Description Sequence SEQ ID No. AACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCCC CAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACACGTC CACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACT CTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGCGGTGGAT
CTCCGAAAAAGAAGAGAAAGGTGTAATGA Sequence of MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS 3 amino acids GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE Cas9 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKADLRLIYLALAHMI GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDG
GGSPKKKRKV AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCG 4 open reading GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA mRNA from GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA Cas9 (ORF) 2 GCACUGCUGUUCGACAAGAGGAGAAACCAGA mRNA GGSPKKKRKV
Description Sequence SEQ ID No. GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA
Description Sequence SEQ ID No. AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC
Description Sequence SEQ ID No. UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG
GAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG Cas9 mRNA AUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAAUUCCGUGG 5 ORF 1 GUUGGGCAGUGAUCACGGAUGAAUACAAAGUGCCGUCCAAGAAGUUCAA GGUCCUGGGGAACACCGAUAGACACAGCAUCAAGAAAAAUCUCAUCGGA GCCCUGCUGUUUGACUCCGGCGAAACCGCAGAAGCGACCCGGCUCAAAC GUACCGCGAGGCGACGCUACACCCCGGCGGAAGAAUCGCAUCUGCUAUCU GCAAGAGAUCUUUUCGAACGAAAUGGCAAAGGUCGACGACAGCUUCUUC CACCGCCUGGAAGAAUCUUUCCUGGUGGAGGAGGACAAGAAGCAUGAAC GGCAUCCUAUCUUUGGAAACAUCGUCGACGAAGUGGCGUACCACGAAAA GUACCCGACCAUCUACCAUCUGCGGAAGAAGUUGGUUGACUCAAACUGAC AAGGCCGACCUCAGAUUGAUCUACUUGGCCCUCGCCCAUAUGAUCAAAU UCCGCGGACACUUCCUGAUCGAAGGCGAUCUGAACCCUGAUAACUCCGA CGUGGAUAAGCUUUUCAUUCAACUGGUGCAGACCUACAACCAACUGUUC GAAGAAAACCCAAUCAAUGCUAGCGGCGUCGAUGCCAAGGCCAUCCUGU CCGCCCGGCUGUCGAAGUCGCGGCGCCUCGAAAACCUGAUCGCACAGCU GCCGGGAGAGAAAAAGAACGGACUUUUCGGCAACUUGAUCGCUCUCUCA CUGGGACUCACUCCCAAUUUCAAGUCCAAUUUGACCUGGCCGAGGACG CGAAGCUGCAACUCUCAAAGGACACCUACGACGACGACUUGGACAAUUU GCUGGCACAAAUUGGCGAUCAGUACGCGGAUCUGUUCCUUGCCGCUAAG AACCUUUCGGACGCAAUCUUGCUGUCCGAUAUCCUGCGCGUGAACACCG AAAUAACCAAAGCGCCGCUUAGCGCCUCGAUGAUUAAGCGGUACGACGA GCAUCACCAGGAUCUCACGCUGCUCAAAGCGCUCGUGAGACAGCAACUG CCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAAUGGGUACG CAGGGUACAUCGAUGGAGGCGCUAGCCAGGAAGAGUUCUAUAAGUUCAU CAAGCCAAUCCUGGAAAAAGAUGGACGGAACCGAAGAACUGCUGGUCAAG CUGAACAGGGAGGAUCUGCUCCGGAAACAGAGAACCUUUGACAACGGAU CCAUUCCCCACCAGAUCCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCG CCAGGAGGACUUUUACCCAUUCCUCAAGGACAACCGGGAAAAGAUCGAG AAAAUUCUGACGUUCCGCAUCCCGUAUUACGUGGGCCCACUGGCGCGCG GCAAUUCGCGCUUCGCGUGGAUGACUAGAAAAUCAGAGGAAAACCAUCAC UCCUUGGAAUUUCGAGGAAGUUGUGGAUAAGGGAGCUUCGGCACAAAGC
Description Sequence SEQ ID No. UUCAUCGAACGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGU GCUUCCUAAGCACAGCCUCCUUUACGAAUACUUCACUGUCUACAACGAAC UGACUAAAGUGAAAUACGUUACUGAAGGAAUGAGGAAGCCGGCCUUUCU GUCCGGAGAACAGAAGAAAGCAAUUGUCGAUCUGCUGUUCAAAGACCAAC CGCAAGGUGACCGUCAAGCAGCUUAAAGAGGACUACUUCAAGAAGAUCG AGUGUUUCGACUCAGUGGAAAUCAGCGGGGUGGAGGACAGAAUUCAACGC UUCGCUGGGAACCUAUCAUGAUCUCCUGAAGAUCAUCAAGGACAAGGAC UUCCUUGACAACGAGGAGAACGAGGACAUCCUGGAAGAUAUCGUCCUGA CCUUGACCCUUUUCGAGGAUCGCGAGAUGAUCGAGGAGAGGCUUAAGAC CUACGCUCAUCUCUUCGACGAUAAGGUCAUGAAACAACUCAAGCGCCGC CGGUACACUGGUUGGGGCCGCCUCUCCCGCAAGCUGAUCAACGGUAUUC GCGAUAAACAGAGCGGUAAAACUAUCCUGGAUUUCCUCAAAUCGGAUGG CUUCGCUAAUCGUAACUUCAUGCAAUUGAUCCACGACGACGACAGCCUGACC UUUAAGGAGGACAUCCAAAAAAGCACAAGUGUCCGGACAGGGAGACUCAC UCCAUGAACACAUCGCGAAUCUGGCCGGUUCGCCGGCGAUUAAGAAGGG AAUUCUGCAAACUGUGAAGGUGGUCGACGAGCUGGUGAAGGUCAUGGGA CGGCACAAACCGGAGAAUCGUGAUUGAAAUGGCCCGAGAAAACCAGAC UACCCAGAAGGGCCAGAAAAACUCCCGCGAAAGGAUGAAGCGGAUCGAA GAAGGAAUCAAGGAGCUGGGCAGCCAGAUCCUGAAAGAGCACCCGGUGG AAAAACACGCAGCUGCAGAACGAGAAGCUCUACCUGUACUAUUUGCAAAAU GGACGGGACAUGUACGUGGACCAAGAGCUGGACAUCAAUCGGUUGUCUG AUUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGGAUGACUC GAUCGAUAACAAGGUGUUGACUCGCAGCGACAAGAACAGAGGGAAGUCA GAUAAUGUGCCAUCGGAGGAGGUCGUGAAGAAGAUGAAGAAUUACUGGC GGCAGCUCCUGAAUGCGAAGCUGAUUACCCAGAGAAAGUUUGACAAUCU CACUAAAGCCGAGCGCGGCGGACUCUCAGAGCUGGAUAAGGCUGGAUUC AUCAAACGGCAGCUGGUCGAGACUCGGCAGAUUACCAAGCACGUGGCGC AGAUCUUGGACUCCCGCAUGAACACUAAAUACGACGAGAACGAUAAGCUC AUCCGGGAAGUGAAGGUGAUUACCCUGAAAAGCAAACUUGUGUCGGACU UUCGGAAGGACUUUCAGUUUUACAAAGUGAGAGAAAUCAACAACUACCAU CACGCGCAUGACGCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCA AAAAAGUACCCUAAACUUGAAUCGGAGUUUGUGUACGGAGACUACAAGGU CUACGACGUGAGGAAGAUGAUAGCCAAGUCCGAACAGGAAAUCGGGAAA GCAACUGCGAAAUACUUCUUUUACUCAAACAUCAUGAACUUUUUCAAGAC UGAAAUUACGCUGGCCAAUGGAGAAAUCAGGAAGAGGCCACUGAUCGAA ACUAACGGAGAAACGGGCGAAAUCGUUGGGGACAAGGGCAGGGACUUCG CAACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCAAUAUUGUGAAGAAA
Description Sequence SEQ ID No. ACCGAAGUGGCAAACCGGCGGAUUUUCAAAGGAAUCGAUCCUCCCAAAGA GAAAUAGCGACAAGCUCAUUGCACGCAAGAAAGACUGGGACCCGAAGAAG UACGGAGGAUUCGAUUCGCCGACUGUCGCAUACUCCGUCCUCGUGGUG GCCAAGGUGGAGAAGGGAAAGAGCAAAAAGCUCAAAUCCGUCAAAGAGCU GCUGGGGAUUACCAUCAUGGAACGAUCCUCGUUCGAGAAGAACCCGAUU GAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUGAAGAAGGAUCUGAUCA UCAAACUCCCCAAGUACUCACUGUUCGAACUGGAAAUGGUCGGAAGCG CAUGCUGGCUUCGGCCGGAGAACUCCAAAAGGAAAUGAGCUGGCCUUG CCUAGCAAGUACGUCAACUUCCUCUAUCUUGCUUCGCACUACGAAAAACU CAAAGGGUCACCGGAAGAUACGAACAGAAGCAGCUUUCGUGGAGCAG CACAAGCAAUUAUCUGGAUGAAAUCAUCGAACAAAUCUCCGAGUUUUCAAAA GCGCGUGAUCCUCGCCGACGCCAACCUCGACAAAAGUCCUGUCGGCCUAC AAUAAGCAUAGAGAUAAGCCGAUCAGAGAACAGGCCGAGAACAUUAUCCA CUUGUUCACCCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUACUUC GAUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAAGUUCUGGA CGCGACCCUGAUCCACCAAAGCAUCACUGGACUCUACGAAACUAGGAUC GAUCUGUCGCAGCUGGGUGGCGAUGGCGGUGGAUCUCCCAAAAAGAAGA
GAAAGGUGUAAUGA MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS 6 amino acid sequence GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE cas9 nickase DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR (D10A) GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA
Description Sequence SEQ ID No. HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDG
GGSPKKKRKV ORF of AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG 7 mRNA of GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA cas9 nickase GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA (D10A) GCACUGCUGUUCGACAGCGGAGAAAACAACAGAGA GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU
Description Sequence SEQ ID No. UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU
Description Sequence SEQ ID No. CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG
GAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG Sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS 8 amino acids GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE dCas9 (D10A DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR H840A) GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA
Description Sequence SEQ ID No. HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDG
GGSPKKKRKV ORF of AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG 9 mRNA of GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA dCas9 (D10A GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA H840A) GCACUGCUGUUCGACAGCGGAGACAGACAGCGGAGAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA H840A)GCACUGCUGUUCGACAGCGGAGACAGACAGCGGAGAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA H840A)GGSPKKKKRKV ORF of AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG. GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU
Description Sequence SEQ ID No. UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU
Description Sequence SEQ ID No. CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG
GAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG Sequence of GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAU 10 encoding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU naked Cas9 CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAAACAGCAGAAGCAACAAGACUGAAGAGAACA GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGGCGACGUCGACAAG CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAG CUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCACAGA UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA
Description Sequence SEQ ID No. CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUGAUCAAGAGAUACGACGAACACCACCAGGAC CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU
Description Sequence SEQ ID No. GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG
GAGGAAGCCCGAAGAAGAAGAGAAAGGUC Sequence of GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAU 11 coding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU naked cas9 CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC nickase UGCUGUUCGACAGCGGAGAAACAGCAGAGAGACAGACAGACAGACAGACAGACAGACAGAGACAGACAGAGAGACAGACAGAGAGACAGACAGAGAGACAGACAGAGAGAGACAGACAGAGAGACAGACAGACAGAGACAGACAGACAGACAGAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG
Description Sequence SEQ ID No. AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGGCGACGUCGACAAG CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAG CUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCACAGA UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUGAUCAAGAGAUACGACGAACACCACCAGGAC CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU
Description Sequence SEQ ID No. GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA
Description Sequence SEQ ID No. CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG
GAGGAAGCCCGAAGAAGAAGAGAAAGGUC sequence of GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAU 12 coding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU naked dCas9 CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAAACAGCAGAAGCAACAAGACUGAAGAGAACA GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGGCGACGUCGACAAG CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAG CUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCACAGA UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUGAUCAAGAGAUACGACGAACACCACCAGGAC CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG
Description Sequence SEQ ID No. CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA
Description Sequence SEQ ID No. GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG
GAGGAAGCCCGAAGAAGAAGAGAAAGGUC Sequence MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS 13 GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE cas9 of amino acids (without DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR NLS) GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT
Description Sequence SEQ ID No.
LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD ORF mRNA AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCG 14 GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA cas9 GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA that encodes GCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGA SEQ ID NO: 13 GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG using codons CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC minimum ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC uridine CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA as listed in Table 3 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG with ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA start codon and stop GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC
Description Sequence SEQ ID No. GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA
Description Sequence SEQ ID No. GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA
GGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACUAG Sequence GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAU 15 coding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU cas9 that CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC encoding SEQ UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACA ID NO: 13 GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA using AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC codons UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG minimum AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC uridine AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC as UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA listed in CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAG Table 3 (without CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC codons GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG beginning or AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA stopped, adequate AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGAC CUGGCAGAAGACGCAAAGCUGCAG CUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGA for inclusion in the coding sequence CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGAC the fusion protein) CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU
Description Sequence SEQ ID No. CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA
Description Sequence SEQ ID No. AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA
GGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS sequence of 16 amino acids of GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE cas9 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR nickase (GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR without NLS) RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD
Description Sequence SEQ ID No. MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT
LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD ORF mRNA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG 17 GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA nickase cas9 GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA GCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGA encoding SEQ ID NO: 16 using GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA codons CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC minimum ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC uridine CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA as listed in Table 3 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG with ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA start codon and stop GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU
Description Sequence SEQ ID No. UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA
Description Sequence SEQ ID No. GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA
GGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACUAG Sequence GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAU 18 coding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU CS9 Nickase CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC encoding UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACA SEQ ID NO: GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA 16 using AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC codons UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG minimum AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC uridine AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC as UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA listed in CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAG Table 3 (without CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC codons GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG beginning or AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA stopped, adequate AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAA CUUCGACCUGGCAGAAGACGCAAAGCUGCAG for inclusion CUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCACAGA
Description Sequence SEQ ID No. in the sequence UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA encoding CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG of fusion proteins CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGAC fusion proteins) CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG
Description Sequence SEQ ID No. AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA
GGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC Sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS 19 GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE amino acid dCas9 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR (without NLS) GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE
Description Sequence SEQ ID No. KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT
LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD ORF AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG 20 dCas9 GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA of mRNA encoding GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA GCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGA SEQ ID NO: 19 GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA using codons CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC minimum ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC uridine CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA as listed in Table 3 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG with ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA start codon and stop GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA
Description Sequence SEQ ID No. AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC
Description Sequence SEQ ID No. AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA
21 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAU GGACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACUAG sequence encoding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU dCas9 that CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACA encoding SEQ ID NO: 19 GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC using codons UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG minimum AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC uridine AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC as UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA
Description Sequence SEQ ID NO CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAG listed in Table 3 (without CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG start codon or stop AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA; AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAG suitable for inclusion in CUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGA CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG coding sequence of UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGAC fusion proteins) CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC
Description Sequence SEQ ID No. AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAG GAGGAAGC
Description Sequence SEQ ID NO: 22 amino acid sequence of MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE of cas9 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR with two signal GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL nuclear location as amino acids C-terminal DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
ORF 23 GSGSPKKKRKVDGSPKKKRKVDSG AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCG mRNA encoding cas9 GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA SEQ ID NO: 22 GCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGA GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG using codons CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA minimum CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC uridine ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC as listed in AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA
Description Sequence SEQ ID No. Table 3, with GGACACUUCCUGAUCGAAGGAGAGACCUGAACCCGGACAACAGCGACGUCG codons ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA start and AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA stop GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA
Description Sequence SEQ ID No. ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAA GCGGAAGCCCGAAGAAGAAGAGAAAAGGUCGACGGAAGCCCGAAGAAGAA
Description Sequence SEQ ID No.
GAGAAAGGUCGACAGCGGAUAG Sequence GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAGCGUCGGAU 24 encoding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU cas9 that CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC encoding SEQ UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACA ID NO: 23 GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA using AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC codons UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG minimum AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC uridine AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC as UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA listed in CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAG Table 3 (without CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC codons GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG beginning or AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA stopped, adequate AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAG to inclusion CUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGA in the UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA coding CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG of the CACCGCUGAGCGCAAGCAUGAUCAAGAUACGACGAACACCACCAGGAC merge) AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG
Description Sequence SEQ ID No. YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA
Description Sequence SEQ ID No. GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAA GCGGAAGCCCGAAGAAGAAGAGAAAAGGUCGACGGAAGCCCGAAGAAGAA
GAGAAAGGUCGACAGCGGA MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS sequence of 25 amino acids of GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE cas9 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR nickase GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR with two signal RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD nuclear location as amino acids C-terminal LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL
Description Sequence SEQ ID No. FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGS
GSPKKKRKVDGSPKKKRKVDSG ORF mRNA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG 26 GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA cas9 nickase GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA GCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGA encoding SEQ ID NO: 25 using GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA codons CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC minimum ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC uridine CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA as listed in Table 3 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG with ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA start codon and stop GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG
Description Sequence SEQ ID No. AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA
Description Sequence SEQ ID No. GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAA GCGGAAGCCCGAAGAAGAAGAGAAAAGGUCGACGGAAGCCCGAAGAAGAA
GAGAAAGGUCGACAGCGGAUAG Sequence GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAU 27 coding GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU cas9 nickase CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC encoding UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACA SEQ ID NO: GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA 25 using AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC codons UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG minimum AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC uridine AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC as UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA listed in CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAG Table 3 (without CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC codons GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG beginning or AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA stopped, adequate AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAG CUGCAG for inclusion CUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGA in the UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA coding CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG of CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGAC fusion proteins) AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC
Description Sequence SEQ ID No. GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACCACAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA
Description Sequence SEQ ID No. GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGA
AGAAGAGAAAGGUCGACAGCGGA Sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS 28 amino acids of GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE dCas9 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR two signals GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD nuclear location as amino acids C-terminal LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD
Description Sequence SEQ ID No. FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYYLQNGRD MYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGS
GSPKKKRKVDGSPKKKRKVDSG ORF mRNA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCG 29 GAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAA dCas9 GGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGA that encodes GCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGA SEQ ID NO: 28 GAACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG using codons CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCA CAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGAC minimum ACCCGAUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUAC uridine CCGACAAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGC AGACCUGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA as listed in Table 3 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCG with ACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAA AACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAA start codon and stop GACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGG AGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCU GCAGCUGAGCAAGGACACAAUACGACGACGACCUGGACAACCUGCUGGCA CAGAUCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGA GCGACGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACA AAGGCACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCCACCA GGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAG
Description Sequence SEQ ID No. UACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAU CGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUC CUGGAAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAG AAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACU UCUACCCGUUCCUGAAGGACAACAGAGAAAAAAGAUCGAAAGAUCCUGACA UUCAGAAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAU UCGCAUGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUC GAAGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAA UGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCAC AGCCUGCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAA GUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAG AAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGC GUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACAU ACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAA GAAAACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGA AGACAGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGG AAGACUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAA AGACAAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUC AUGCAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAA GGCACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAAC CUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGG UCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAU CGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGA ACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGA AGCCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGA AAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGAC CAGGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCG UCCCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAG UCGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCU GAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGAC UGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAAC AAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACA CAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA
Description Sequence SEQ ID No. CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACA AGGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAAC GCAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCG AAUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGC AAAGAGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAA AUCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGU CUGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUG CCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCA GCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAG UCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAA GAAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGA AGCAGCUUCGAAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGA
AGAAGAGAAAGGUCGACAGCGGAUAG sequence encoding GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAGCGUCGGAU 30 GGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAAGAAGUUCAAGGU dCas9 that CCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAUCGGAGCAC UGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACA encoding SEQ ID NO: 28 GCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA using codons AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGAC UGGAAGAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCG minimum AUCUUCGGAAACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGAC uridine AAUCUACCACCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC as UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGAGGACA
Description Sequence SEQ ID NO CUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAGCGACGUCGACAAG listed in Table 3 (without CUGUUCAUCCAGCUGGUCCAGACAUACAACCAGCUGUUCGAAGAAAACCC GAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCUGAGCGCAAGACUG start codon or stop AGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA; AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUGAC ACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAG suitable for inclusion in CUGAGCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGA CGCAAUCCUGCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG sequence encoding the UCGGAGACCAGUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGA CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCACCAGGAC fusion protein) CUGACACUGCUGAAGGCACUGGUCAGACAGCAGCUGCCGGAAAAGUACA AGGAAAAUCUUCUUCGACCAGAGCAAGAACGGAUACGCAGGAUACAUCGAC GGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGG AAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGAC CUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCACCAGAU CCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUAC CCGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAG AAUCCCGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCA UGGAUGACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGA AGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACA AACUUCGACAAGAACCUGCCGAACGAAAAGGUCCUGCCGAAGCACAGCCU GCUGUACGAAUACUUCACAGUCUACAACGAACUGACAAAGGUCAAGUACG UCACAGAAGGAAUGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAA GGCAAUCGUCGACCUGCUGUUCAAGACAAAACAGAAAGGUCACAGUCAAG CAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUCGACAGCGUCG YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYED GACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAAA CGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGAC AGAGAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGA CAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGA CUGAGCAGAAAGCUGAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGAC AAUCCUGGACUUCCUGAAGAGCGACGGAUUCGCAAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGG CACAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAAACCU GGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAGACAGUCAAGGUC GUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGAAAACAUCG UCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAAC
Description Sequence SEQ ID No. AGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAA AGCUGUACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCA GGAACUGGACAUCAACAGACUGAGCGACUACGACGUCGACGCAAUCGUC CCGCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUCCUGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGU CGUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAAGCUG AUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAGAGAGAGGAGGACU GAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAGCUGGUCGAAACA AGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAAUGAACAC AAAGUACGAACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAA GGUCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACG CAGUCGUCGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGA AUUCGUCUACGGAGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCA AAGAGGCGAACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAACGGAGAAA UCAGAAAGAGACCGCUGAUCGAAACAAACGGAGAAAACAGGAGAAAUCGUC UGGGACAAGGGAAGAGACUUCGCAACAGUCAGAAAGGUCCUGAGCAUGC CGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAG CAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGAA AGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCGACAGU CGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAAGAGCAAG AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAA GCAGCUUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAA GGAAGUCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGC AGAAGGGAAACGAACUGGCACUGCCGAGCAAGUACGUCAACUUCCUGUA CCUGGCAAGCCACUACGAAAAGCUGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCUGUUCGUCGAACAGCACAAGCACUACCUGGACGAAAUCAU CGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAACC UGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGCCGAUCAGA GAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGGAGC ACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACA CAAGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACA GGACUGUACGAAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAGCCCGAAGA
Description Sequence SEQ ID No.
AGAAGAGAAAGGUCGACAGCGGA T7 promoter TAATACGACTCACTATA 31 beta-globin ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC 32 5' UTR human beta-globin GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTC 33 3'UTR CAACTACTAAACTGGGGGATATTATGAAGGGAGCCTTG
CCTAATAAAAAACATTTATTTTCATTGC alpha-globin 5 'UTR CATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGGTCCCCACAGAC 34 TCAGAGAGAACCCACC human alpha-globin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCC 35 3'UTR human CCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA GTGGGCGGC Xenopus laevis AAGCTCAGAATAAACGCTCAACTTTGGCC 36 beta-globin 5'
Xenopus UTR ACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTT 37 laevis beta- ACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTA globin 3' ATAAAAAGAAAGTTTCTTCACATTCT
UTR growth hormone of bovine CAGGGTCCTGTGGACAGCTCACCAGCT 38 5'UTR Hormone TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGA 39 AGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA bovine growth 3'UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTG 40 Hemoglobin alpha Mus musculus CACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG, adult chain 1 (HBA-A1), HSD17B4 3'UTR 5'TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGC 41
UTR AGGCCTTATTC Guide RNA mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAm 42 G282 AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm
Description Sequence SEQ ID NO directed to TTR AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC mU mU * * mu * 43 mU Transcribed GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT cas9 with 5 'UTR TGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGAC HSD TGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATAC
ORF correspondent AAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAG CATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAG CAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAA and SEQ ID NO: 4 AGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGG TCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAG Kozak sequence and 3 'UTR ACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG ALB CATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACAC ATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGAC AACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAG CTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAAGGCAAT CCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCAC AGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAAGAGCAACTTCGACCTGGCAGAAGA CGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACC TGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAG AACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAA ATCACAAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACA CCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGG AAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGAT ACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGA TCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGA GAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCA CCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACT TCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTC GCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAA GAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGAC AAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGT CACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG
Description Sequence SEQ ID No. CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGAC ATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATG ATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATG AAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAA GCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTT CCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGA CGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGAC AGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCA ATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAG AAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAG AGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACA CCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGAC TGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGAC GACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAA GAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACT GGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAAC CTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATT CATCAAGAGACAGCTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACA GATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGAT CAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAG AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGC ACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTA CCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGT CAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAA AGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACT GGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAA CAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAG GTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGAC AGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGC TGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG AAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCAT
Description Sequence SEQ ID No. GGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGG GATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCC TGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA CTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCT GTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCA TCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACC TGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGA GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCA CCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGG ACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAAGGTCTAGCTAGCCATCACATTTAAAAAGCA TCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATT CATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACAT AAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATG
GAAAGAACCTCGAG Transcript GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTCGT 44 Cas9 with 5' TGCAGGCCTTATTCGGATCCATGGACAAGAAGTACAGCATCGGACTGGACA UTR from HSD, TCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTC
ORF correspondent CCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAA GAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAG CAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACA and SEQ ID NO: 4 and 3 'UTR GAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACG ALB ACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGA AGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACC ACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCA CAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCA AGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGC GACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTC GAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAG CGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGCTGC CGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTG GGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAA GCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGG CACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG
Description Sequence SEQ ID No. AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACA AAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGT ACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCG ACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGG AAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGAC CTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGAT CCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACC CGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAA TCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGG ATGACAAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTC GTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTC GACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTA CGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGA AGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCG TCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAGG AAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAG TCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGA TCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGG AAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAG AAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGC TGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATC AACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAA GAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACA GCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGA GACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAA GAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCA TGGGAAGACACAAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAACC AGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATC GAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAA CGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCG ACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGA CAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGAC AGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAA AGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAG
Description Sequence SEQ ID No. AGACAGCTGGTCGAAACAAAGACAGATCACAAAGCACGTCGCACAGATCCTG GACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAA GTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACCGCACACGAC GCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAA GCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAA GATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTT CTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAAC GGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAAACAGGAGA AATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGA GCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGA TTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGC AAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGA CAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGC AAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGA AGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAG GAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAA CTGGAAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAA GGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGG CAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAG CAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATCGAACAG ATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAG GTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGGC AGAAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCAGC ATTCAAGTACTTCGACACAACAATCGACAGAAAAGATACACAAGCACAAA GGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACG AAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCC GAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGC CTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCT TTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTC TTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGA
ACCTCGAG ORF of Cas9 ATGGATAAGAAGTACTCGATCGGGCTGGATATCGGAACTAATTCCGTGGGT 45 alternative TGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTC with U content of CTGGGGAACACCGATAGACACAGCATCAAGAAGAATCTCATCGGAGCCCT 19.36% GCTGTTTGACTCCGGCGAAACCACCGATAGAGACACAGCATCAAGAAGAATCTCATCGGAGCCCT 19.36% GCTGTTTGACTCCGGCGAAACCACCGATAGAGACACAGCATCAAGAAGAATCTCATCGGAGCCCT 19.36% GCTGTTTGACTCCGGCGAAACCCCAGTTCAAGGTCGAAACCACCGAGGTCGAAACCACCGAGGTCGAAACCACCGAGGCCGAAACCACCGAGACCTCGAG ORF by Cas9
Description Sequence SEQ ID No. CGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAA ATCTTTTCGAACGAAATGGCAAAGGTGGACGACAGCTTCTTCCACCGCCTG GAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATC TTTGGAAACATCGTGGACGAAGTGGCGTACCACGAAAAGTACCCGACCATC TACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGA TTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGA TCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTC AACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAAACCCAATCAATGCCA GCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCG GCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAGAAGAACGGAC TTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTC CAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTA CGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGA TCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATC CTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATT AAGCGGTACGACGAGCATCACCAGGATTCCACGCTGCTCAAAGCGCTCGT GAGACAGCAACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAA GAATGGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCT ATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGC TGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACAGAGAACCTTTGACA ACGGAAGCATTCCACACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGC GGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATC GAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCG CGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCAC TCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAATCCTT CATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCT TCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACT AAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGA GAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTG ACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGAC TCAGTGGAAATCAGCGGAGTGGAGGACAGATTCAACGCTTCGCTGGGAAC CTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGA GGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGA GGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGA CGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCC GCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAA CTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCA
Description Sequence SEQ ID No. GTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACA AGTGAGCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCG GTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGTGGTGGAC GAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGA AATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCG AAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATC CTGAAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTA CCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGA CATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTT CTGAAGGATGACTCCATCGATAACAAGGTGTTGACTCGCAGCGACAAGAAC AGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAA GAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTT TGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGG CTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCAC GTGGCGCAGATCCTGGACTCCCGCATGAACACTAAATACGACGAGAACGAT AAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCG GACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACC ATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCCTGATC AAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTC TACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAGC AACTGGCAAATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGACTGAAA TTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACG GAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTT CGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTG CAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGAC AAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATT CGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGA AGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACC ATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTGGAGGCG AAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTGCCCAAGTAC TCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGG AGAACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTT CCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGATAA CGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAAT CATCGAACAAATCTCCGAGTTTTCAAAAGCGCGTGATCTCGCCGACGCCAA CCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAG AGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGC
Description Sequence SEQ ID No. TCCAGCCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACAC GTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTG GACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATGGTGGC GGTGGATCCTACCCATACGACGTGCCTGACTACGCCTCCGGAGGTGGTGG
CCCCAAGAAGAAACGGAAGGTGTGATAG cas9 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 46 with TGCAGGCCTTATTCGGATCTGCCACCATGGATAAGAAGTACTCGATCGGGC transcript 5 'UTR of TGGATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACA HSD ORF AAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGC Corresponding to ATCAAGAAGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCA GAAGCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGA SEQ ID NO: 45, AGAATCGCATCTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGT GGACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGG Kozak sequence, and ACAAGAAGCATGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGG 3'UTR of CGTACCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTG ALB ACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATA TGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATA ACTCCGACGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAAC TGTTCGAAGAAAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCC TGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAG CTGCCGGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCA CTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCG AAGCTGCCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTG GCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTT TCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACC AAAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCA GGATTCCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTA CAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGA TGGAGGCGCCAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGA AAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATC TGCTCCGCAAACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCC ATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCA TTCCTCAAGGACAACCGGGAAAAGATCGAGAAAAATCTGACGTTCCGCATC CCGTATTACGTGGGCCCACTGGCGCGGCAATTCGCGCTTCGCGTGGAT GACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGT GGATAAGGGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGA
Description Sequence SEQ ID No. CAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGA ATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGA ATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGA TCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGA CTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGA GGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCAT CAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAG ATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGA GGCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAA GCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACG GTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAAATCGGA TGGCTTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAGCCTGAC CTTTAAGGAGGACATCCAGAAAGCACAAGTGAGCGGACAGGGAGACTCAC TCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGA ATCCTGCAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACG GCAAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTAC CCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAG GAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAAC ACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGG GACATGTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGAC GTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAAC AAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCC ATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGA ATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGC GCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTG GTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCG CATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGT GATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTT TACAAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTC AACGCTGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCG GAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCC AAGTCCCGAACAGGAAATCGGGAAAGCAACTGGCAAATACTTCTTTTACTCAA ACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAG GAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGTGGG ACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAG TCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAAT CGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACT
Description Sequence SEQ ID No. GGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCC GTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCTCAAATC CGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAA GAACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGG ATCTGATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCG GAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAGGAAATGAGCTGG CCTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAGAA ACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCA GCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAG CGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAA TAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTT GTTCACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAGTACTTCGATAC TACTATCGACCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGAC CCTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTC GCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCTG ACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAGGTGTGATAG CTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGA AAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGC CAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCT
GTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Transcript of GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 47 Cas9 with 5' TGCAGGCCTTATTCGGATCTATGGATAAGAAGTACTCGATCGGGCTGGATA UTR from HSD, TCGGAACTAATTCCGTGGTTGGGCAGTGATCACGGATGAATACAAAGTGC
ORF correspondent CGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCATCAAGA AGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCG ACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCG and SEQ ID NO: 45 and 3 'UTR CATCTGCTATCTGCAAGAAATCTTTTCGAACGAAATGGCAAAGGTGGACGA ALB CAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAA GCATGAACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTACCA CGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAAC TGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAA ATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGA CGTGGATAAGCTGTTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGA AGAAAACCCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCG CCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCG GGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGA
Description Sequence SEQ ID No. CTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTG CAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAA ATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGAC GCAATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCG CCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTC ACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGA GATTTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAGG CGCCAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGAT GGACGGAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCC GCAAACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGG GTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCA AGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATT ACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGA AAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAG GGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAAT CTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTC ACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGG AAGCCGGCCTTTCTGAGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCT GTTCAAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTT CAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACA GATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGG ACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCG TCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTA AGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCC GCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATT CGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAAATCGGATGGC TTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAGCCTGACCTTTA AGGAGGACATCCAGAAAGCACAAGTGAGCGGACAGGGAGACTCACTCCAT GAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCT GCAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACA AACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCAGA AGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATC AAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCA GCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACAT GTACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGA CCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAACAAGGT GTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGG
Description Sequence SEQ ID No. AGGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCG AAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGC GGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGA GACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGA ACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTA CCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAA AGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGC TGTGGTCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTT TGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTC CGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATC ATGAACTTCTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAAG AGGCCACTGATCGAAACTAACGGAGAAACGGGGCAAATCGTGTGGGACAA GGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAA TATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGAT CCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGA CCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTCCT CGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCA AAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACC CGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTG ATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAG CGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAGGAAATGAGCTGGCCTT GCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAGAAACTC AAAGGGTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCA CAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGC GTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAG CATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTC ACCCTGACTAACCTGGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACT ATCGACCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCT GATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCA GCTGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCTGACT ACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAGGTGTGATAGCTA GCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAA TGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAA CACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGT
GCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Transcript of GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 48
Description Sequence SEQ ID NO cas9 TGCAGGCCTTATTCGGATCCATGCCTAAGAAAAAGCGGAAGGTCGACGGG compreenden GATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGG the ORF GCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTCCTG cas9 GGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTG using codons with TTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAG expression GCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATCTT TTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCCTGGAAGA generally high in human ATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCTTTGG AAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCA TCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGATTGAT CTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCGA AGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAACT GGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGG CGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCC TCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCG GCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTT TGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACG ACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTGT TCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGC GCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAGC GGTACGACGAGCATCACCAGGATTCCACGCTGCTCAAAGCGCTCGTGAGA CAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAAT GGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAA GTTCATCAAGCCAATCCTGGAAAAAGATGGACGGAACCGAAGAACTGCTGGT CAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACG GATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGC GCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGA AAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGC AATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCT TGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATC GAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCT AAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAG TGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAAC AGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCG TCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAG TGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTAT CATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAG
Description Sequence SEQ ID No. AACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGAT CGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGAT AAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCT CTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAACTAT CCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTG ATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTG TCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTC GCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGC TGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAATG GCCCGAGAAAACCAGACTACCCCAGAAGGGCCAGAAAAACTCCCGCGAAAG GATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGA AAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCTG TACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATC AATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTGA AGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAG GGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATT ACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACA ATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGA TTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGC GCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCT CATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTT TCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCAC GCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAA GTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGA CGTGAGGAAGATGATAGCCAAGTCCCGAACAGGAAATCGGGAAAGCAACTG CGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAATTACG CTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGA AACGGGGCAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCA AAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAACCGAAGTGCAAA CCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGC TCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATT CGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGGA AAGAGCAAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATG GAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGGGT TACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTACTCACTG TTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACT CCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTA
Description Sequence SEQ ID No. TCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACA GAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGA ACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGA CAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAACA GGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCCCCAGC CGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACACGTCCACC AAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTAC GAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGATAGTCTAGCCAT CACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAG ATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCC TGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCA
ATTAATAAAAAATGGAAAGAACCTCGAG transcript GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 49 cas9 TGCAGGCCTTATTCGGATCCGCCACCATGCCTAAGAAAAAGCGGAAGGTC compreenden GACGGGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTG of the GGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAG GTCCTGGGGAACACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCC Kozak sequence with ORF CTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTAC cas9 CGCGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAG AGATCTTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACCGCC using codons with TGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTA TCTTTGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCA generally high expression in human TCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCA GATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCT GATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCAT TCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCCAATCAATGC TAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGC GGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGA CTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGT CCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCT ACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGG ATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATAT CCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGA TTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTC GTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCC AAGAATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTT
Description Sequence SEQ ID No. CTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACT GCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTG ACAACGGATCCATTCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCT TGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAG ATCGAGAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCG CGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATC ACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGC TTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTG CTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGA CTAAAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCG GAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGG TGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCG ACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGA ACCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACG AGGAGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCG AGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCG ACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGC CGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAA ACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGC AATTTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCAC AAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCC GGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGA CGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTG AAATGGCCCGAGAAAACCAGACTACCCCAGAAGGGCCAGAAAAACTCCCGC GAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGAT CCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCT ACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTGG ACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTT TCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAA CAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGA AGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGT TTCAAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAG GCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCA CGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGA TAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTC GGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTAC CATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGAT
Description Sequence SEQ ID No. CAAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGT CTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAAAG CAACTGGCAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAA ATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAAC GGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGT TCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGT GCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAATAGCGA CAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGAT TCGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAG AAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGCTGCTGGGGATTACC ATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCG AAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCAAGTAC TCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGG AGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTT CCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAA CGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAAT CATCGAACAAATCTCCGAGTTTTCAAAAGCGCGTGATCTCGCCGACGCCAA CCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAG AGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGGGAGC CCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGATACAC GTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTG GACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGATAGT CTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGA AAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGC CAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCT
GTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGG 50 ORF with ATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGT CCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCAC splices removed; TGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACA 12.75% of GCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAA U content ATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGG AAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCT TCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCT ACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGA CTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTG
Description Sequence SEQ ID No. ATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGC AAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCA GAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGA CTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAG AGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACAC ATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACG CAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGC GACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAG CATGATCAAAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGG CACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACC AGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAA GAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAA GAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAAC ATTCCAAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACG CAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAG AAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGC TGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAA ACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGC ACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGA AAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAA CGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATT CCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAA ACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCG AATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCC TGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGA CACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCAC ACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAG GATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAG AGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAAACAG AAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACAT CCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCG CAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTC AAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAA CATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGG
Description Sequence SEQ ID No.
GAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAAC GAAAAGCTGTACCTGTACTACCTGCAaAACGGAAGAGACATGTACGTCGAC CAGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAA GAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTC GTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATC ACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAG CGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGAC AGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGT ACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGA GCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAG AAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCG GAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACG GAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAG GAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACT TCTTCAAGACAGAAATCACACTGGCAAACGGAGAAAATCAGAAAGAGACCGC TGATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGA GACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGA AGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTC GTCGCAAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGA ACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGAT CGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCAT CAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAAGGAAGAAAGAGAAT GCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGA GCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGG GAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAG CACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTC ATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCA CAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCAC ACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAAT CGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGAT CCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCT
GGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAG Transcript of GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 51
Description Sequence SEQ ID No. Cas9 with 5' TGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGAC UTR of HSD, TGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATAC
ORF correspondent AAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAG CATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAG CAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAA and SEQ ID NO: 50, AGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGG TCGACGACAGCTTCTTCCACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGA Kozak sequence and 3 'UTR CAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG ALB CATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACAC ATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGAC AACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAG CTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAAGGCAAT CCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCAC AGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAAGAGCAACTTCGACCTGGCAGAAGA CGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACC TGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAG AACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAA ATCACAAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACA CCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGG AAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGAT ACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGA TCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGA GAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCA CCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACT TCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTC GCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAA GAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGAC AAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGT CACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGAC
Description Sequence SEQ ID No. ATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATG ATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATG AAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAA GCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTT CCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGA CGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGAC AGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCA ATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAG AAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAG AGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACA
CCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACT GAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACG ACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAG AGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTG GAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCT GACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCA TCAAGAGACAGCTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGA TCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCA GAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAA AGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCAC ACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACC CGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCA GAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAG TACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGG CAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACA GGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGT CCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAG GAGGATTCAAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTG ATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAG CCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAA AGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATG GAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGG ATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCT GTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAAC TGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGT
Description Sequence SEQ ID No. ACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAA CAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATC GAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGA ACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACC GGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAG CACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACT GTACGAAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA GCCCGAAGAAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCT CAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCAT CTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAA TTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATTGGAA
AGAACCTCGAG ORF cas9 ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGG 52 with codons CTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGG minimum TGCTGGGCAACACCGACAGACACAGCATCAAGAAGAACCTGATCGGCGCC uridine CTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCAGACTGAAGAGAAC CGCCAGAAGAAGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGA be frequently used in humans GATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACT you generally GGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACACCCC; 12.75% ATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCAC of U content CATCTACCACCTGAGAAAGAAGCTGGTGGACAGCACCGACAAGGCCGACC TGAGACTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACT TCCTGATCGAGGGGCACCTGAACCCCGACAACAGCGACGTGGACAAGCTG TTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATC AACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCAGACTGAGCA AGAGCAGAAGACTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAG AACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAA CTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCA AGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGGCAC CAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCT GCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGA GCGCCAGCATGATCAAAGATACGACGAGCACCACCAGGACCTGACCCTG CTGAAGGCCCTGGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTT CTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCA GCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGAC
Description Sequence SEQ ID No. GGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGACCTGCTGAGAAA GCAGAGAACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCG AGCTGCACGCCATCCTGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAG GACAAACAGAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACTAC GTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAAA GAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGG GCGCCAGCGCCCAGAGCTTCATCGAGAGAATGACCAACTTCGACAAGAAC CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTT CACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGA GAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTG CTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGAGGACTA CTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGG ACAGATTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGAC ATCGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAG ACTGAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAA GAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAGAAAGCTGATCAACG GCATCAGAGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGC GACGGCTTCGCCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGGCAC AGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAA GGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATG GGCCAGACACAAGCCCGAGAACATCGTGATCGAGATGGCCAGAGAGAACCA GACCACCCAGAAGGGCCAGAAGAACAGCAGAGAGAGAATGAAGAGAATCG AGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGT GGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGA ACGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAGACTGAGC GACTACGACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAG CATCCAAACAAGGTGCTGACCAGAAGCGACAAGAACAGAGGCAAGAGCG ACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGA CAGCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACC AAGGCCGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCA AGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCACGTGGCCCAGATC CTGGACAGCAGAATGAACACCAAGTACGACGAGAACGACAAGCTGATCAG AGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAA AGGACTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCACGCCC ACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTAC
Description Sequence SEQ ID No. CCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGT GAGAAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCA AGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCT GGCCAACGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGA CCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGAAAG GTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGAC CGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGAGAAACAGCGACAAGC TGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGAC AGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGG GCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATC ATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAA GGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACA GCCTGTTCGAGCTGGAGAACGGCAGAAAGAGAATGCTGGCCAGCGCCGG CGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGAC AACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGA GATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTGATCCTGGCCGACG CCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCC ATCAGAGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTG GGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAG ATACACCAGCACCAAGGAGGTGCTGGACGCCACCCCTGATCCACCAGAGCA TCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAGCTGGGCGGCGAC
GGCGGCGGCAGCCCCAAGAAGAAGAGAAAGGTGTGA Transcript of GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 53 Cas9 with 5' TGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGC UTR from HSD, CTGGACATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTA
ORF correspondent CAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACA GCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACC GCCGAGGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAGAAG and SEQ ID NO: 52, AAAGAACAGAATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAA GGTGGACGACAGCTTCTTCCACAGACTGGAGGAGAGCTTCCTGGTGGAGG Kozak sequence and 3 'UTR AGGACAAGAAGCACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAG ALB GTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAGCTG GTGGACAGCCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTGGC CCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGGCACCTGAACCC CGACAAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACA
Description Sequence SEQ ID No. ACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAG GCCATCCTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTGAT CGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCG CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCC GAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGA CAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCG CCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAAC ACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAGCAGC TGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTAC GCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGC TGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGC ATCCCCCACCAGATCCACCTGGGGCAGCTGCACGCCATCCTGAGAAGACA GGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAGATCGAGAAGAT CCTGACCTTCAGAATCCCCTACTACGTGGGCCCCCTGGCCAGAGGCAACA GCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAGAGACCATCACCCCCTGG AACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGA GAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCA AGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGG TGAAGTACGTGACCGAGGGCATGAGAAAGCCCGCCTTCCTGAGCGGCGAG CAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACAGAAAGGTGAC CGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACA GCGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCAC CTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA GGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCG AGGACAGAGAGATGATCGAGGAGAGACTGAAGACCTACGCCCACCTGTTC GACGACAAGGTGATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGG CAGACTGAGCAGAAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCA AGACCATCCTGGACTTCCTGAAGAGGCGACGGCTTCGCCAAACAGAAACTTCA TGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAG GCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACC TGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTG GTGGACGAGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCGT GATCGAGATGGCCAGAGAGAACCAGACCACCAGAAGGGCCAGAAGAACA GCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGAGCTGGGCAG CCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGA
Description Sequence SEQ ID No. AGCTGTACCTGTACTACCTGCAGAACGGCAGAGACATGTACGTGGACCAG GAGCTGGACATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCC CCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAA GCGCAAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCCAAGCTGATCAC CCAGAGAAAGTTCGACAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCG AGCTGGACAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAG ATCACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTAC GACGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTGAGAGA GATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGG GCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTAC GGCGACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCA GGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAA CTTCTTCAAGACCGAGATCACCTGGCCAACGGCGAGATCAGAAAGAGAC CCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGG CAGAGACTTCGCCACCGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACA TCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATC CTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGA CCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGC TGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGT GAAGGAGCTGCTGGGCATCACCATCATGGAGAGAAGCAGCTTCGAGAAGA ACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGAC CTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAG AAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTG GCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGA GAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTG GAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTT CAGCAAGAGAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCG CCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAACATC ATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTA CTTCGACACCACCATCGACAGAAAGAGATACACCAGCACCAAGGAGGTGCT GGACGCCACCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCAGAA TCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAA GAGAAAGGTGTGACTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGA GAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTT CGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATT
Description Sequence SEQ ID No.
TTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG ORF cas9 ATGGACAAAAAATACAGCATAGGGCTAGACATAGGGACGAACAGCGTAGG 54 with codons GTGGGCGGTAATAACGGACGAATACAAAGTACCGAGCAAAAAATTCAAAGT minimum ACTAGGGAACACGGACCGACACAGCATAAAAAAAAACCTAATAGGGGCGCT uridine rarely ACTATTCGACAGCGGGGAAACGGCGGAAGCGACGCGACTAAAACGAACGG CGCGACGACGATACACGCGACGAAAAAACCGAATATGCTACCTACAAGAAA used in humans TATTCAGCAACGAAATGGCGAAAGTAGACGACAGCTTCTTCCACCGACTAG generally AAGAAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAACGACACCCGATAT; 12.75% TCGGGAACATAGTAGACGAAGTAGCGTACCACGAAAAATACCCGACGATAT of U content ACCACCTACGAAAAAAACTAGTAGACAGCACGGACAAAGCGGACCTACGAC TAATATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGGCACTTCCTAAT AGAAGGGGACCTAAACCCGGACAACAGCGACGTAGACAAACTATTCATACA ACTAGTACAAACGTACAACCAACTATTCGAAGAAAACCCGATAAACGCGAG CGGGGTAGACGCGAAAGCGATACTAAGCGCGCGACTAAGCAAAAGCCGAC GACTAGAAAACCTAATAGCGCAACTACCGGGGGAAAAAAAAACGGGCTAT TCGGGAACCTAATAGCGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCA ACTTCGACCTAGCGGAAGACGCGAAACTACAACTAAGCAAAGACACGTACG ACGACGACCTAGACAACCTACTAGCGCAAATAGGGGACCAATACGCGGAC CTATTCCTAGCGGCGAAAAACCTAAGCGACGCGATACTACTAAGCGACATA CTACGAGTAAACACGGAAATAACGAAAGCGCCGCTAAGCGCGAGCATGATA AAACGATACGACGAACACCACCAAGACCTAACGCTACTAAAAGCGCTAGTA CCAAACAACTACCGGAAAAATACAAAAGAAATATTCTTCGACCAAAGCAAAA ACGGGTACGCGGGGTACATAGACGGGGGGGCGAGCCAAGAAGAATTCTAC AAATTCATAAAACCGATACTAGAAAAAATGGACGGGACGGAAGAACTACTA GTAAAACTAACCGAGAAGACCTACTACGAAAACAACGAACGTTCGACAAC GGGAGCATACCGCACCAAATACACCTAGGGGAACTACACGCGATACTACG ACGACAAGAAGACTTCTACCCGTTCCTAAAAGACAACCGAGAAAAAATAGA AAAAAATACTAACGTTCCGAATACCGTACTACGTAGGGCCGCTAGCGCGAGG GAACAGCCGATTCGCGTGGATGACGCGAAAAAGCGAAGAAACGATAACGC CGTGGAACTTCGAAGAAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCTTC ATAGAACGAATGACGAACTTCGACAAAAACCTACCGAACGAAAAAAGTACTA CCGAAACACAGCCTACTATACGAATACTTCACGGTATACAACGAACTAACGA AAGTAAAATACGTAACGGAAGGGATGCGAAAACCGGCGTTCCTAAGCGGG GAACAAAAAAAAGCGATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAA CGGTAAAACAACTAAAAGAAGACTACTTCAAAAAAATAGAATGCTTCGACAG CGTAGAAATAAGCGGGGTAGAAGACCGATTCAACGCGAGCCTAGGGACGT
Description Sequence SEQ ID No. ACCACGACCTACTAAAAATAATAAAAGACAAAGACTTCCTAGACAACGAAGA AAACGAAGACATACTAGAAGACATAGTACTAACGCTAACGCTATTCGAAGAC CGAGAAATGATAGAAGAACGACTAAAAACGTACGCGCACCTATTCGACGAC AAAGTAATGAAACAACTAAAACGACGACGATACACGGGGTGGGGGCGACT AAGCCGAAAACTAATAAACGGGATACGAGACAAAAAAAGCGGGAAAACGAT ACTAGACTTCCTAAAAAGGCGACGGGTTCGCGAACCGAAACTTCATGCAACT AATACACGACGACAGCCTAACGTTCAAAGAAGACATACAAAAGCGCAAGT AAGCGGGCAAGGGGACAGCCTACACGAACACATAGCGAACCTAGCGGGGA GCCCGGCGATAAAAAAAAGGGATACTACAAACGGTAAAAGTAGTAGACGAAC TAGTAAAAGTAATGGGGCGACACAAACCGGAAAACATAGTAATAGAAATGG CGCGAGAAAACCAAACGACGCAAAAAGGGCAAAAAAACAGCCGAGAACGA ATGAAACGAATAGAAGAAGGGATAAAAGAACTAGGGAGCCAAATACTAAAA GAACACCCGGTAGAAAAACACGCAACTACAAAACGAAAAACTATACCTATACT ACCTACAAAACGGGCGAGACATGTACGTAGACCAAGAACTAGACATAAACC GACTAAGCGACTACGACGTAGACCACATAGTACCGCAAAGCTTCCTAAAAG ACGACAGCATAGACAACAAAGTACTAACGCGAAGCGACAAAAACCGAGGG AAAAGCGACAACGTACCGAGCGAAGAAGTAGTAAAAAAAATGAAAAAACTAC TGGCGACAACTACTAAACGCGAAACTAATAACGCAACGAAAATTCGACAAC CTAACGAAAGCGGAACGAGGGGGGCTAAGCGAACTAGACAAAGCGGGGTT CATAAAACGACAACTAGTAGAAACGCGACAAATAACGAAACACGTAGCGCA AATACTAGACAGCCGAATGAACACGAAATACGACGAAAACGACAAACTAAT ACGAGAAGTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGACTTCCG AAAAGACTTCCAATTCTACAAAGTACGAGAAATAAACAACTACCCACCACGCG CACGACGCGTACCTAAACGCGGTAGTAGGGACGGCGCTAATAAAAAAAATAC CCGAAACTAGAAAGCGAATTCGTATACGGGGACTACAAAGTATACGACGTA CGAAAAATGATAGCGAAAAGCGAACAAGAAATAGGGAAAGCGACGGCGAA ATACTTCTTCTACAGCAACATAATGAACTTCTTCAAAAGGAAATAACGCTA GCGAACGGGGAAATACGAAAACGACCGCTAATAGAAACGAACGGGGAAAC GGGGGAAATAGTATGGGACAAAGGGGCGAGACTTCGCGACGGTACGAAAAG TACTAAGCATGCCGCAAGTAAACATAGTAAAAAAAAACGGAAGTACAAACGG GGGGGTTCAGCAAAGAAAGCATACTACCGAAACGAAACAGCGACAAACTAA TAGCGCGAAAAAAAGACTGGGACCCGAAAAAATACGGGGGGTTCGACAGC CCGACGGTAGCGTACAGCGTACTAGTAGTAGCGAAAGTAGAAAAAAGGGAA AAGCAAAAAACTAAAAAGCGTAAAAGAACTACTAGGGATAACGATAATGGAA CGAAGCAGCTTCGAAAAAAACCCGATAGACTTCCTAGAAGCGAAAGGGTAC AAAGAAGTAAAAAAAGACCTAATAATAAAACTACCGAAATACAGCCTATTCG AACTAGAAAACGGGCGAAAACGAATGCTAGCGAGCGCGGGGGAACTACAA
Description Sequence SEQ ID No. AAAGGGAACGAACTAGCGCTACCGAGCAAATACGTAAACTTCCTATACCTA GCGAGCCACTACGAAAAAACTAAAGGGAGCCCGGAAGACAACGAACAAAA ACAACTATTCGTAGAACAAACACAAACACTACCTAGACGAAATAATAGAACAA ATAAGCGAATTCAGCAAACGAGTAATACTAGCGGACGCGAACCTAGACAAA GTACTAAGCGCGTACAACAAACACCGAGACAAACCGATACGAGAACAAGCG GAAAACATAATACACCTATTCACGCTAACGAACCTAGGGGCGCCGGCGGC GTTCAAATACTTCGACACGACGATAGACCGAAAAACGATACACGAGCACGAA AGAAGTACTAGACGCGACGCTAATACACCAAAGCATAACGGGGCTATACGA AACGCGAATAGACCTAAGCCAACTAGGGGGGGACGGGGGGGGGAGCCCG
AAAAAAAAAACGAAAAGTATGA Transcript of GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 55 Cas9 with 5' TGCAGGCCTTATTCGGATCCGCCACCATGGACAAAAAATACAGCATAGGGC UTR from HSD, TAGACATAGGGACGAACAGCGTAGGGTGGGCGGTAATAACGGACGAATAC
ORF correspondent AAAGTACCGAGCAAAAAATTCAAAGTACTAGGGAACACGGACCGACACAGC ATAAAAAAAAACCTAATAGGGGCGCTACTATTCGACAGCGGGGAAACGGCG GAAGCGACGCGACTAAAACGAACGGCGCGACGACGATACACGCGACGAAA and SEQ ID NO: 54, AAACCGAATATGCTACCTACAAGAAATATTCAGCAACGAAATGGCGAAAGTA GACGACAGCTTCTTCCACCGACTAGAAGAAAGCTTCCTAGTAGAAGAAGAC Kozak sequence and 3 'UTR AAAAAACACGAACGACACCCGATATTCGGGAACATAGTAGACGAAGTAGCG ALB TACCACGAAAAATACCCGACGATATACCACCTACGAAAAAAACTAGTAGACA GCACGGACAAAGCGGACCTACGACTAATATACCTAGCGCTAGCGCACATGA TAAAATTCCGAGGGCACTTCCTAATAGAAGGGGACCTAAACCCGGACAACA GCGACGTAGACAAACTATTCATACAACTAGTACAAACGTACAACCAACTATT CGAAGAAAACCCGATAAACGCGAGCGGGGTAGACGCGAAAGCGATACTAA GCGCGCGACTAAGCAAAAGCCGACGACTAGAAAACCTAATAGCGCAACTAC CGGGGGAAAAAAAAAAACGGGCTATTCGGGAACCTAATAGCGCTAAGCCTA GGGCTAACGCCGAACTTCAAAAGCAACTTCGACCTAGCGGAAGACGCGAA ACTACAACTAAGCAAAGACACGTACGACGACGACCTAGACAACCTACTAGC GCAAATAGGGGACCAATACGCGGACCTATTCCTAGCGGCGAAAAACCTAAG CGACGCGATACTACTAAGCGACATACTACGAGTAAACACGGAAATAACGAA AGCGCCGCTAAGCGCGAGCATGATAAAACGATACGACGAACACCCACCAAG ACCTAACGCTACTAAAAAGCGCTAGTACGACAACAACTACCGGAAAAAATACA AAGAAATATTCTTCGACCAAAGCAAAAACGGGTACGCGGGGTACATAGACG GGGGGGCGAGCCAAGAAGAATTCTACAAATTCATAAAACCGATACTAGAAA AAATGGACGGGACGGAAGAACTACTAGTAAAACTAAACCGAGAAGACCTAC TACGAAAACAACGAACGTTCGACAACGGGAGCATACCGCACCAAATACACC
Description Sequence SEQ ID No. TAGGGGAACTACACGCGATACTACGACGACAAGAAGACTTCTACCCGTTCC TAAAAGACAACCGAGAAAAAATAGAAAAAATACTAACGTTCCGAATACCGTA CTACGTAGGGCCGCTAGCGCGAGGGAACAGCCGATTCGCGTGGATGACG CGAAAAAGCGAAGAAACGATAACGCCGTGGAACTTCGAAGAAGTAGTAGAC AAAGGGGCGAGCGCGCAAAGCTTCATAGAACGAATGACGAACTTCGACAA AAACCTACCGAACGAAAAAAGTACTACCGAAACACAGCCTACTATACGAATAC TTCACGGTATACAACGAACTAACGAAAGTAAAATACGTAACGGAAGGGATG CGAAAACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCGATAGTAGACCTA CTATTCAAAACGAACCGAAAAGTAACGGTAAAACAACTAAAAGAAGACTACT TCAAAAAAATAGAATGCTTCGACAGCGTAGAAATAAGCGGGGTAGAAGACC GATTCAACGCGAGCCTAGGGACGTACCACGACCCTACTAAAAATAATAAAAG ACAAAGACTTCCTAGACAACGAAGAAAACGAAGACATACTAGAAGACATAG TACTAACGCTAACGCTATTCGAAGACGAGAAATGATAGAAGAACGACTAA AAACGTACGCGCACCTATTCGACGACAAAGTAATGAAACAACTAAAACGAC GACGATACACGGGGTGGGGGCGACTAAGCCGAAAACTAATAAACGGGATA CGAGACAAAACAAAGCGGGAAAACGATACTAGACTTCCTAAAAAGCCGACGGG TTCGCGAACCGAAACTTCATGCAACTAATACACGACGACAGCCTAACGTTC AAAGAAGACATACAAAAAAGCGCAAGTAAGCGGGCAAGGGGACAGCCTACA CGAACACATAGCGAACCTAGCGGGGAGCCCGGCGATAAAAAAGGGATAC TACAAACGGTAAAAGTAGTAGACGAACTAGTAAAAGTAATGGGGCGACACA AACCGGAAAACATAGTAATAGAAATGGCGCGAGAAAACCAAAACGACGCAAA AAGGGCAAAAAAACAGCCGAGAACGAATGAAACGAATAGAAGAAGGGATAA AAGAACTAGGGAGCCAAATACTAAAAAGAACACCCGGTAGAAAAACACGCAAC TACAAAACGAAAAAACTATACCTATACTACCTACAAAACGGGCGAGACATGTA CGTAGACCAAGAACTAGACATAAACCGACTAAGCGACTACGACGTAGACCA CATAGTACCGCAAAGCTTCCTAAAAAGACGACAGCATAGACAACAAAGTACT AACGCGAAGCGACAAAAACCGAGGGAAAAGCGACAACGTACCGAGCGAAG AAGTAGTAAAAAAAATGAAAAACTGGCGACAACTACTAAACGCGAAACT AATAACGCAACGAAAATTCGACAACCTAACGAAAGCGGAACGAGGGGGGC TAAGCGAACTAGACAAAGCGGGGTTCATAAAACGACAACTAGTAGAAAACGC GACAAATAACGAAACACGTAGCGCAAATACTAGACAGCCGAATGAACACGA AATACGACGAAAACGACAAACTAATACGAGAAGTAAAAGTAATAACGCTAAA AAGCAAACTAGTAAGCGACTTCCGAAAAGACTTCCAATTCTACAAAGTACGA GAAATAAACAACTACCACCACGCGCACGACGCGTACCTAAACGCGGTAGTA GGGACGGCGCTAATAAAAAAAATACCCGAAACTAGAAAGCGAATTCGTATAC GGGGACTACAAAGTATACGACGTACGAAAAATGATAGCGAAAAGCCGAACAA GAAATAGGGAAAGCGACGGGCAAATACTTCTTCTACAGCAACATAATGAAC
Description Sequence SEQ ID No. TTCTTCAAAACGGAAATAACGCTAGCGAACGGGGAAATACGAAAACGACCG CTAATAGAAACGAACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGCG AGACTTCGCGACGGTACGAAAAGTAACTAAGCATGCCGCAAGTAAACATAGT AAAAAAAAACGGAAGTACAAACGGGGGGGTTCAGCAAAGAAAGCATACTACC GAAACGAAACAGCGACAAACTAATAGGCGCAAAAAAAGACTGGGACCCGAA AAAATACGGGGGGTTCGACAGCCCGACGGTAGCGTACAGCGTACTAGTAG TAGCGAAAGTAGAAAAAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAAAGAAC TACTAGGGATAACGATAATGGAACGAAGCAGCTTCGAAAAAAACCCGATAG ACTTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAAAGACCTAATAATAAAAA ACTACCGAAATACAGCCTATTCGAACTAGAAAACGGGCGAAAACGAATGCT AGCGAGCGCGGGGGAACTACAAAAAGGGAACGAACTAGCGCTACCGAGCA AATACGTAAACTTCCTATACCTAGCGAGCCACTACGAAAAACTAAAGGGA GCCCGGAAGACAACGAACAAAAACAACTATTCGTAGAACAACACAAACACT ACCTAGACGAAATAATAGAACAAATAAGCGAATTCAGCAAACGAGTAATACT AGCGGACGCGAACCTAGACAAAGTACTAAGCGCGTACAACAAACACCGAG ACAAACCGATACGAGAACAAGCGGAAAACATAATACACCTATTCACGCTAA CGAACCTAGGGGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAGAC CGAAAACGATACACGAGCACGAAAGAAGTACTAGACGCGACGCTAATACAC CAAAGCATAACGGGGCTATACGAAACGCGAATAGACCTAAGCCAACTAGGG GGGGACGGGGGGGGGGAGCCCGAAAAAAAAAACGAAAAGTATGACTAGCCAT CACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAG ATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCC TGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCA
ATTAATAAAAAATGGAAAGAACCTCGAG transcript AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 56 cas9 with TGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGAC as primary AGG TGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATAC AAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAG three nucleotides for use with CATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAG CAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAA CleanCapTM, AGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGG 5 'UTR TCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAG HSD ACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG ORF correspondent CATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACAC and SEQ ID NO: 4 ATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGAC
Description Sequence SEQ ID No. Sequence of AACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAG Kozak and 3' CTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAAT UTR of ALB CCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCAC AGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAAGAGCAACTTCGACCTGGCAGAAGA CGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACC TGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAG AACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAA ATCACAAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACA CCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGG AAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGAT ACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGA TCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGA GAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCA CCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACT TCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTC GCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAA GAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGAC AAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGT CACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGAC ATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATG ATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATG AAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAA GCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTT CCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGA CGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGAC AGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCA ATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAG AAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAG AGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACA
Description Sequence SEQ ID No. CCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGAC TGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGAC GACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAA GAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACT GGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAAC CTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATT CATCAAGAGACAGCTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACA GATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGAT CAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAG AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGC ACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTA CCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGT CAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAA AGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACT GGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAA CAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAG GTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGAC AGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGC TGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG AAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCAT GGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGG GATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCC TGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA CTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCT GTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCA TCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACC TGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGA GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCA CCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGG ACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAAGGTCTAGCTAGCCATCACATTTAAAAAGCA TCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATT CATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACAT
Description Sequence SEQ ID No. AAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATG
GAAAGAACCTCGAG Transcript of GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAG 57 Cas9 with 5' ACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACG CMV UTR, CGGATTCCCCGTGCCAAGAGTGACTCACCGTCCTTGACACGGCCACCATG
ORF correspondent GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATG GGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGC and SEQ ID NO: 4 TGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCA AGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATC Kozak sequence and 3 'UTR TTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA ALB GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTC GGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTAC CACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACT GATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGAT CGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCC AGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAA GCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGA AGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACT GTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGA GCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGC AGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCG ACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCA TGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCA CTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGA ATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGA ACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATT CGACAAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAA TCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAA AGATCGAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGG CAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAA TCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAG AGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAG GTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAA
Description Sequence SEQ ID No. CTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAG AAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATG CTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGA CAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACT GTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACACCT GTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGAT GGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGC GGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAAC TTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAG AAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAA CCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGG TCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATC GTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAA CAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAA GCCAGATCCTGAAGGAACACCCCGGTCGAAAACACACAGCTGCAGAACGAA AAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAG GAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCC GCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAA GCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTC AAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACA CAGAGAAAGTTCGACAACCCTGACAAAAGGCAGAGAGAGGAGGACTGAGCGA ACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGAT CACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGA CGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAA GCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAAT CAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAA CAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAA TCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACT TCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAG AGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAAGAA GTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCG
Description Sequence SEQ ID No. CAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTG CTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGAC TTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAG CTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTG GCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAA GTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAG CCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACT ACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGA GACAAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTG ACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGAC AGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCAC CAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGG AGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAAGGTCTAGCTAGCCA TCACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAA GATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACC CTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTC
AATTAATAAAAAATGGAAAGAACCTCGAG Transcript of GGGacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccggatctgccaccATGGA 58 Cas9 with 5' CAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG UTR from HBB, CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAG
ORF correspondent GAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGT TCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGA AGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTC and SEQ ID NO: 4 AGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAA AGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGA Kozak sequence and 3 'UTR AACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCAC HBB CTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGAT CTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGA AGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGC TGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCG GAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGA CTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTT CGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCA ACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATAC GACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGA
Description Sequence SEQ ID No. CCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACA TCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGA TCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTG GTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGC AAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATT CTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACT GCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCG ACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATC CTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAG ATCGAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCA AGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATC ACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAG CTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGT CCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACT GACAAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGA GCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGA AAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGC TTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGA CAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACT GTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACACCT GTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGAT GGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGC GGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAAC TTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAG AAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAA CCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGG TCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATC GTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAA CAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAA GCCAGATCCTGAAGGAACACCCCGGTCGAAAACACACAGCTGCAGAACGAA AAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAG GAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCC GCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAA GCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTC AAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACA CAGAGAAAGTTCGACAACCCTGACAAAAGGCAGAGAGAGGAGGACTGAGCGA
Description Sequence SEQ ID No. ACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGAT CACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGA CGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAA GCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAAT CAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAA CAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAA TCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTT CAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGAT CGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACT TCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAG AGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAAGAA GTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCG CAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTG CTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGAC TTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAG CTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTG GCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAA GTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAG CCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACT ACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGA GACAAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTG ACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGAC AGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCAC
CAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGG AGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGctagcgctcg ctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggc cttgagcatctggattctgcctaataaaaaacatttattttcattgcctcgag transcript GGGaagctcagaataaacgctcaactttggccggatctgccacCATGGACAAGAAGTACAGCA 59 cas9 with 5 'UTR TCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGAC XBG, GAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAG
ORF ACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAG corresponding to AAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAA and SEQ ID GAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGG
Description Sequence SEQ ID No. NO: 4, CAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCG sequence from AAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACG Kozak and 3' AAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGC UTR of XBG TGGTCGACAGCACAGACAGACTGCGCAGACCTGAGAGACTGCAGACCTGAGAGACTGCAGACCTGAGAGACTGCAGACCTGAGAGACTGCAGACCTGAGACT GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAAC CCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATA CAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTG ATCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGAT CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGG CAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTG GACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGC AGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAAGAGATACG ACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAG CTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATAC GCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATC AAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCT GAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCA TCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAG GAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATC CTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAG CAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAA CTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAA GAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGC ACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCA AGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAG AAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTC AAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTC GAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCA CGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAA CGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAG AGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGA GCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAAGACAATC CTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTG ATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT
Description Sequence SEQ ID No. CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAA GCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAA CTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATG GCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAG AATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAA GGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTA CTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAA CAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGA AGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGA GGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAA CTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGA CAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAG GATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCG CACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGC TGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACT TCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACC AGCCAACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAG AAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTAC GACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAAC AGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATC ACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGG AGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCA GAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAAGACAGAAGTC CAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGA CAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGAT TCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAA AAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGC AAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTA CAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAG GAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAAC TTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGA CAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACG CAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCG ATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTG GGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGA
Description Sequence SEQ ID No. TACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATC
ACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGG AGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGctagcaccagcctcaagaacac ccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgt atctgctcctaataaaaagaaagtttcttcacattctctcgag transcript AGGaagctcagaataaacgctcaactttggccggatctgccacCATGGACAAGAAGTACAGCA 60 cas9 with TCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGAC as primary AGG GAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAG ACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAG three nucleotides for use with AAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAA GAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGG CleanCapTM, CAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCG 5 'UTR AAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACG XBG, ORF correspondent AAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGC TGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTG GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAAC and SEQ ID NO: 4, s CCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATA sequence of CAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA Kozak and 3' AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTG UTR of XBG ATCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGAT CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGG CAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTG GACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGC AGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAAGAGATACG ACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAG CTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATAC GCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATC AAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCT GAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCA TCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAG GAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATC CTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAG CAGATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAA CTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAA GAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGC
Description Sequence SEQ ID No. ACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCA AGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAG AAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTC AAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTC GAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCA CGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAA CGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAG AGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGA GCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAAGACAATC CTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTG ATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAA GCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAA CTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATG GCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAG AATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAA GGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTA CTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAA CAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGA AGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGA GGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAA CTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGA CAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAG GATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCG CACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGC TGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACT TCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACC AGCCAACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAG AAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTAC GACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAAC AGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATC ACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGG AGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCA GAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAAGACAGAAGTC CAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGA CAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGAT
Description Sequence SEQ ID No. TCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAA AAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCAC AATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGC AAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTA CAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAG GAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAAC TTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGA CAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACG CAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCG ATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTG GGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGA TACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATC
ACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGG AGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGctagcaccagcctcaagaacac ccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgt atctgctcctaataaaaagaaagtttcttcacattctctcgag transcript AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGT 61 cas9 with TGCAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGAC as primary AGG TGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATAC AAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAG three nucleotides for use with CATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAG CAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAA CleanCapTM, AGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGG 5 'UTR TCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAG HSD ACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG ORF correspondent CATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCACAC and SEQ ID NO: 4 sequence ATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGGAC AACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCAG Kozak and 3' CTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAAT UTR of ALB CCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCAC AGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAAGAGCAACTTCGACCTGGCAGAAGA CGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACC TGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAG
Description Sequence SEQ ID No. AACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAA ATCACAAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACA CCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGG AAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGAT ACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGA TCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGA GAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCA CCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACT TCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACAT TCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTC GCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAA GAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGAC AAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGT CACAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGG CAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGC TGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCA GCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGAC ATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATG ATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATG AAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAA GCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTT CCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCACGA CGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGAC AGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCA ATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAG AAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAG AGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACA CCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGAC TGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGAC GACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAA GAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACT GGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAAC CTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATT
Description Sequence SEQ ID No. CATCAAGAGACAGCTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACA GATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGAT CAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAG AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGC ACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTA CCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGT CAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAA AGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACT GGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAA CAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAG GTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGAC AGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGC TGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG AAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCAT GGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGG GATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCC TGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA CTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCCT GTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCA TCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACC TGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGA GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCA CCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGG ACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAG GAAGCCCGAAGAAGAAGAGAAAAGGTCTAGCTAGCCATCACATTTAAAAAGCA TCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATT CATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACAT AAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATG
GAAAGAACCTCGAG Sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAAAAAAAAAAAAAAAA 62 30/30/39 poly- AAAAAAAAAAACCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 63
Description Sequence SEQ ID NO: 100 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA poly-A RNA guide mC mC * * * GUCCAGCGAGGCAAAGGGUUUUAGAGCUAGAAAUAGCAAGU 64 mA G209 UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCmU mU * * * mU U ORF encodes ATGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATC 65 GGAATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAA CCCGATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAG Neisseria meningitidis TCCCGAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCG cas9 GTCAGAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAG ACTGCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACG GACTGATCAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCA CTGGACAGAAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCT GATCAAGCACAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAAACAG CAGACAAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACAC GCACTGCAGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAA GTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCA CACATTCTCGAGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAA GCAGAAGGAATTCGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAA TCGAAACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTC CAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGC AAAGAACACATACACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAA CCTGAGAATCCTGGAACAGGGATCGGAAAAGCCGCTGACAGACACAGAAA GAGCAACACTGATGGACGAACCGTACAGAAAGTCGAAGCTGACATACGCA CAGGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACT GAGATACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGG CATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAG AAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAATCGGAACAGC ATTCTCGCTGTTCAAGACAGACGAAGACATCACAGGAAGACTGAAGGACAG AATCCAGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAA GTTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGA ACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGGAGACCACT ACGGAAAGAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCA GACGAAATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAA GGTCATCAACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCCACA TCGAAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCG AAAAGAGACAGGAAGAAAAACAGAAAGGACAGAGAAAAGGCAGCAGCAAAG TTCAGAGAATACTTCCCGAACTTCGTCGGAGAACCGAAGTCGAAGGACATC
Description Sequence SEQ ID No. CTGAAGCTGAGACTGTACGAACAGCAGCACGGAAAGTGCCTGTACTCGGG AAAGGAAATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGA CCACGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGT CCTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTACG AATACTTCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTCAAGGCAA GAGTCGAAACATCGAGATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGC AGAAGTTCGACGAAGACGGATTCAAGGAAAAGAAACCTGAACGACACAAGAT ACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAG GAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCACAAACCTG CTGAGAGGATTCTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGACA CCACGCACTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGC AGAAGATCACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAA AGACAATCGACAAGGAAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCC CGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTCGGAA AGCCGGACGGAAAGCCGGAATTCGAAGAAGCAGACACACTGGAAAAGCTG AGAACACTGCTGGCAGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGA ATACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGG GACAGGGACACATGGAAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGA GTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGACCTGGA AAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGG CAAGACTGGAAGCACACAAGGACCGACCCGGCAAAGGCATTCGCAGAACCG TTCTACAAGTACGACAAGGCAGGAAACAGAACACACAGCAGGTCAAGGCAGTC AGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGG AATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAGGGAGA CAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCAAAGGGAATCCT GCCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTG ATCGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTC GAAGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCAC AGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAAGATC GGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACTGTCGTT CCAGAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCGTGCAGAC TGAAGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCG
GAATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGA ORF which GCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATCGGA 66 encodes ATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAAGAAGAAAACCC Neisseria GATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCG meningitidis CGAAGACAGGAAAGCGAACGAGACTCAGCAAGACGACTGCAAGACTCA
Description Sequence SEQ ID NO cas9 (no AGAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACT GCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGAC start codons or stop TGATCAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTG; GACAGAAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGAT CAAGCACAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAG suitable for inclusion in ACAAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCA CGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACA coding sequence of CTGCAGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTT CATTCTCGAGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGC fusion proteins) AGAAGGAATTCGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATC GAAACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCA GAAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAA AGAACACATACACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACC TGAGAATCCTGGAACAGGGATCGGAAAAGCCGCTGACAGACACAGAAAGA GCAACACTGATGGACGAACCGTACAGAAAGTCGAAGCTGACATACGCACA GGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGA GATACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGGCA TACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAA GTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAATCGGAACAGCAT TCTCGCTGTTCAAGACAGACGAAGACATCACAGGAAGACTGAAGGACAGAA TCCAGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGT TCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGAAC AGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGGAGACCACTAC GGAAAGAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGA CGAAATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGG TCATCAACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCG AAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGAAA AGAGACAGGAAGAAAAACAGAAAGGACAGAGAAAAGGCAGCAGCAAAGTTC AGAGAATACTTCCCGAACTTCGTCGGAGAACCGAAGTCGAAGGACATCCTG AAGCTGAGACTGTACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAA GGAAATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCA CGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTCCT GGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTACGAAT ACTTCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTCAAGGCAAGA GTCGAAACATCGAGATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAG AAGTTCGACGAAGACGGATTCAAGGAAAAGAAACCTGAACGACACAAGATAC GTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAGGA
Description Sequence SEQ ID No. AAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCACAAACCTGCTG AGAGGATTCTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGACACCA CGCACTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGA AGATCACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGA CAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCCCG CAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTCGGAAAG CCGGACGGAAAGCCGGAATTCGAAGAAGCAGACACACTGGAAAAGCTGAG AACACTGCTGGCAGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAAT ACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGGGA CAGGGACACATGGAAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGT CTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGACCTGGAAA AGATGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGGCA AGACTGGAAGCACACAAGGACGACCCGGCAAAGGCATTCGCAGAACCGTT CTACAAGTACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCA GAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGA ATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAGGGAGAC AAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCAAAGGGAATCCTG CCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTGAT CGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGA AGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAG AGGAACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAAGATCGG AAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACTGTCGTTCCA GAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCGTGCAGACTGA AGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGAA
TTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAA transcript GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGATCCGCCACCAT GGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGGACATCGG 67 comprises SEQ ID NO: 65 AATCGCATCGGTCGGATGGGCAATGGTCGAAATCGACGAAGAAGAAAACC (which encodes CGATCAGACTGATCGACCTGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTC CCGAAGACAGGAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGT CAGAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACT Neisseria meningitidis GCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAAACGGAC cas9) TGATCAAGTCGCTGCCGAACACACCGTGGCAGCTGAGAGCAGCAGCACTG GACAGAAAAGCTGACACCGCTGGAATGGTCGGCAGTCCTGCTGCACCTGAT CAAGCACAGAGGATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAG ACAAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCA CTGCAGACAAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACAAGTT
Description Sequence SEQ ID No. CGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGGACTACTCGCACA CATTCTCGAGAAAGGACCTGCAGGCAGAACTGATCCTGCTGTTCGAAAAGC AGAAGGAATTCGGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATC GAAACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCA GAAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAGCAA AGAACACATACACAGCAGAAAGATTCATCTGGCTGACAAAGCTGAACAACC TGAGAATCCTGGAACAGGGATCGGAAAAGCCGCTGACAGACACAGAAAGA GCAACACTGATGGACGAACCGTACAGAAAGTCGAAGCTGACATACGCACA GGCAAGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGA GATACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGGCA TACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGAAGGACAAGAA GTCGCCGCTGAACCTGTCGCCGGAACTGCAGGACGAAATCGGAACAGCAT TCTCGCTGTTCAAGACAGACGAAGACATCACAGGAAGACTGAAGGACAGAA TCCAGCCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGT TCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGAAC AGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGGAGACCACTAC GGAAAGAAGAACACAGAAGAAAAGATCTACCTGCCGCCGATCCCGGCAGA CGAAATCAGAAACCCGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGG TCATCAACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCG AAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGAAA AGAGACAGGAAGAAAAACAGAAAGGACAGAGAAAAGGCAGCAGCAAAGTTC AGAGAATACTTCCCGAACTTCGTCGGAGAACCGAAGTCGAAGGACATCCTG AAGCTGAGACTGTACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAA GGAAATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCA CGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTCCT GGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACACCGTACGAAT ACTTCAACGGAAAGGACAACTCGAGAGAATGGCAGGAATTCAAGGCAAGA GTCGAAACATCGAGATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAG AAGTTCGACGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAAGATAC GTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAGGA AAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCACAAACCTGCTG AGAGGATTCTGGGGACTGAGAAAGGTCAGAGCAGAAAACGACAGACACCA CGCACTGGACGCAGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGA AGATCACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGA CAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCCCG CAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGTCTTCGGAAAG CCGGACGGAAAGCCGGAATTCGAAGAAGCAGACACACTGGAAAAGCTGAG
Description Sequence SEQ ID No. AACACTGCTGGCAGAAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAAT ACGTCACACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGGGA CAGGGACACATGGAAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGT CTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGACCTGGAAA AGATGGTCAACAGAGAAAGAGAACCGAAGCTGTACGAAGCACTGAAGGCA AGACTGGAAGCACACAAGGACGACCCGGCAAAGGCATTCGCAGAACCGTT CTACAAGTACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCA GAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGA ATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAGGGAGAC AAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCGCAAAGGGAATCCTG CCGGACAGAGCAGTCGTCCAGGGAAAGGACGAAGAAGACTGGCAGCTGAT CGACGACTCGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGA AGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAG AGGAACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAAGATCGG AAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGACAGCACTGTCGTTCCA GAAGTACCAGATCGACGAACTGGGAAAGGAAATCAGACCGTGCAGACTGA AGAAGAGACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGAA TTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGATAGCTAGCTCGAGTC TAGAGGGCCCGTTTAAACCCGCTGATCAGCCCTGACTGTGCCTTCTAGTTG CCAGCCATCTGTTGTTTGCCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA GGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC
TCTATGG Sequence MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGD 68 amino acids of Neisseria meningitidis SLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTP WQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFE cas9 KQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAK NTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKL LGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPE LQDEIGTAFSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPL MEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVIN GVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFP NFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTW DDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRS KKQRILLQKFDEDGFKERNNDTRYVNRFLCQFVADRMRLTGKGKKRVFASN
Description Sequence SEQ ID No. GQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMN AFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLE KLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGV SVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYD KAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLV PIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARM FGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRP
CRLKKRPPVRSGKRTADGSEFESPKKKRKVE RNA mG tab * mc * mc * GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAm 69 G390 AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC mU * mu * mu * mA guide RNA mU * mc * mA * CAAAUACCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAm 70 G502 AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC mU * mu * mu * mU RNA guide mA * mA * mA * GUUCUAGAUGCCGUCCGGUUUUAGAmGmCmUmAmGmAmAm 71 mU AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm G509 * mU mU * * * mU RNA guide mA mC * mG * 72 CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAm G534 AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC mU mU * * * mU mU Sequence TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCG 73 GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG DNA encoding eGFP TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATG CGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATAC CGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCA GGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTA CGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAAC GCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTA ATACGACTCACTATAGGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTT CGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCATGGTGAGCAAGGGCG AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGA CGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC
Description Sequence SEQ ID No. CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCT TCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCA TGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGC AACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAA CCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGG GGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGG AGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG TAATAGGAATTATGCAGTCTAGCCATCACATTTAAAAGCATCTCAGCCTACC ATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCT TTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAAT CATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCG AGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTA GACTTAAGCTTGATGAGCTCTAGCTTGGCGTAATCATGGTCATAGCTGTTTC CTGTGTGAAATTGTTATCCGCTCCAAATTCCACACAACATACGAGCCGGAA GCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAA TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGG GCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAG GCCAGGAACCGTAAAAAAGGCCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCA GTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATT AGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC TAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAA GCCAGTTACCTTCGGAAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG
Description Sequence SEQ ID No. AAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG CTGAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTA TCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGG TGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGAT CAAGGGCAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCA TGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATG CTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACA CGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA TCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATA AACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCT AAGAAACCATTATTATCATGACATTAACCTATAAAAAATAGGCGTATCACGAG
GCCCTTTCGTCG Standard Mn + Mn + Mn + NNNNNNNNNNNNNNNNNGUUUUAG 74 sgRNA AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAA modified AUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm where N is AmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU * mu * mu * mU nucleotides encoding a sequence CMV-1 Chart 5 'UTR CAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT 75 CMV-2 5' UTR AGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGG 76 CMV -3 5' UTR TGCATTGGAACGCGGATTCCCCGTGCCAAAGTGACTCACCG 77
Description Sequence SEQ ID No.
SV40 NLS PKKKRKV 78 NLS one exemplary LAAKRSRTT 79 NLS second exemplary QAAKRSRTT 80 NLS third exemplary PAPAKRERTT 81 NLS fourth exemplary QAAKRPRTT 82 NLS fifth exemplary RAAKRPRTT 83 NLS sixth exemplary AAAKRSWSMAA 84 NLS seventh exemplary AAAKRVWSMAF 85 NLS eighth exemplary AAAKRSWSMAF 86 NLS ninth exemplary AAAKRKYFAA 87 NLS 10 example RAAKRKAFAA 88 NLS 11 example RAAKRKYFAV 89 SV40 alternative NLS PKKKRRV 90 Nucleoplasmin NLS KRPAATKKAGQAKKKK 91 Example encoding sequence for SV40 NLS CCGAAGAAGAAGAGAAAGGTC 92 Example encoding sequence for NLS1 CTGGCAGCAAAGAGAAGCAGAACAACA 92 Example encoding sequence for NLS1 CTGGCAGCAAAGAGAAGCAGAACAACA 92 Example coding sequence for SV40 NLS CCGAAGAAGAAGAGAAAGGTC 92 Example encoding sequence for NLS1 CTGGCAGCAAAGAGAAGCAGAACAACA 92 example RAAKRKAFAA 88 NLS
Description Sequence SEQ ID No.
exemplary coding sequence for NLS2 CAGGCAGCAAAGAGAAGCAGAACAACA 94 exemplary coding sequence for NLS3 CCGGCACCGGCAAAGAGAGAAAGAACAACA 95 exemplary coding sequence for NLS4 CAGGCAGCAAAGAGACCGAGAACAACA 96 exemplary coding sequence for NLS5 AGAGCAGCAAAGAGACCGAGAACAACA 97 exemplary coding sequence for NLS6 GCAGCAGCAAAGAGAAGCTGGAGCATGGCAGCA 98 exemplary coding sequence for NLS7 GCAGCAGCAAAGAGAGTCTGGAGCATGGCATTC 99 Sequence Exemplary coding sequence for NLS8 GCAGCAGCAAAGAGAAGCTGGAGCATGGCATTC 100 Exemplary coding sequence for NLS9 GCAGCAGCAAAGAGAAAGTACTTCGCAGCA 101 Exemplary coding sequence for NLS10 AGAGCAGCAAAGAGAAAGGCATTCGCAGCA 102
Description Sequence SEQ ID NO exemplary coding sequence for NLS11 103 AGAGCAGCAAAGAGAAAGTACTTCGCAGTC exemplary sequence coding for SV40 NLS alternative exemplary CCGAAGAAGAAGAGAAGAGTC 104 Kozak sequence gccgccRccAUGG 105 106 Not used ORF cas9 ATGGACAAGAAGTACTCTATCGGTTTGGACATCGGTACCAACTCTGTCGGTTG using codons half-life of GGCCGTCATCACCGACGAATACAAGGTCCCATCTAAGAAGTTCAAGGTCTTG long GGTAACACCGACAGACACTCTATCAAGAAGAACTTGATCGGTGCCTTGTTGTT CGACTCTGGTGAAACCGCCGAAGCCACCAGATTGAAGAGAACCGCCAGAAGA Table 4 , with AGATACACCAGAAGAAAGAACAGAATCTGCTACTTGCAAGAAATCTTCTCTAA start codons CGAAATGGCCAAGGTCGACGACTCTTTCTTCCACAGATTGGAAGAATCTTTCT and stop TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCAATCTTCGGTAACATCGT CGACGAAGTCGCCTACCACGAAAAGTACCCAACCATCTACCACTTGAGAAAGA AGTTGGTCGACTCTACCGACAAGGCCGACTTGAGATTGATCTACTTGGCCTTG GCCCACATGATCAAGTTCAGAGGTCACTTCTTGATCGAAGGTGACTTGAACCC AGACAACTCTGACGTCGACAAGTTGTTCATCCAATTGGTCCAAACCTACAACC AATTGTTCGAAGAAAACCCAATCAACGCCTCTGGTGTCGACGCCAAGGCCATC TTGTCTGCCAGATTGTCTAAGAGCAGAAGATTGGAAAACTTGATCGCCCAATT GCCAGGTGAAAAGAAGAACGGTTTGTTCGGTAACTTGATCGCCTTGTCTTTGG GTTTGACCCCAAACTTCAAGTCTAACTTCGACTTGGCCGAAGACGCCAAGTTG CAATTGTCTAAGGACACCTACGACGACGACTTGGACAACTTGTTGGCCCAAAT CGGTGACCAATACGCCGACTTGTTCTTGGCCGCCAAGAACTTGTCTGACGCC ATCTTGTTGTCTGACATCTTGAGAGTCAACACCGAAATCACCAAGGCCCCATT GTCTGCCTCTATGATCAAAGATACGACGAACCACCACCAAGACTTGACCTTGT
TGAAGGCCTTGGTCAGACAACAATTGCCAGAAAAGTACAAGGAAATCTTCTTC GACCAATCTAAGAACGGTTACGCCGGTTACATCGACGGTGGTGCCTCTCAAG 107
Description Sequence SEQ ID No. AAGAATTCTACAAGTTCATCAAGCCAATCTTGGAAAAGATGGACGGTACCGAA GAATTGTTGGTCAAGTTGAACAGAGAAGACTTGTTGAGAAAGCAAAGAACCTT CGACAACGGTTCTATCCCACACCAAATCCACTTGGGTGAATTGCACGCCATCT TGAGAAGACAAGAAGACTTCTACCCATTCTTGAAGGACAACAGAGAAAAGATC GAAAAGATCTTGACCTTCAGAATCCCATACTACGTCGGTCCATTGGCCAGAGG TAACAGCAGATTCGCCTGGATGACCAGAAAGTCTGAAGAAACCATCACCCCAT GGAACTTCGAAGAAGTCGTCGACAAGGGTGCCTCTGCCCAATCTTTCATCGAA AGAATGACCAACTTCGACAAGAACTTGCCAAACGAAAAGGTCTTGCCAAAGCA CTCTTTGTTGTACGAATACTTCACCGTCTACAACGAATTGACCAAGGTCAAGTA CGTCACCCGAAGGTATGAGAAAGCCAGCCTTCTTGTCTGGTGAACAAAAGAAG GCCATCGTCGACTTGTTGTTCAAGACCAACAGAAAGGTCACCGTCAAGCAATT GAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACTCTGTCGAAATCTCTG GTGTCGAAGACAGATTCAACGCCTCTTTGGGTACCTACCACGACTTGTTGAAG ATCATCAAGGACAAGGACTTCTTGGACAACGAAGAAAACGAAGACATCTTGGA AGACATCGTCTTGACCTTGACCTTGTTCGAAGACAGAGAAATGATCGAAGAAA GATTGAAGACCTACGCCCACTTGTTCGACGACAAGGTCATGAAGCAATTGAAG AGAAGAAGATACACCGGTTGGGGTAGATTGAGCAGAAAGTTGATCAACGGTA TCAGAGACAAGCAATCTGGTAAGACCATCTTGGACTTCTTGAAGTCTGACGGT TTCGCCAACAGAAACTTCATGCAATTGATCCACGACGACTCTTTGACCTTCAA GGAAGACATCCAAAAGGCCCAAGTCTCTGGTCAAGGTGACTCTTTGCACGAA CACATCGCCAACTTGGCCGGTTCTCCAGCCATCAAGAAGGGTATCTTGCAAAC CGTCAAGGTCGTCGACGAATTGGTCAAGGTCATGGGTAGACACAAGCCAGAA AACATCGTCATCGAAATGGCCAGAGAAAACCAAACCACCCAAAAGGGTCAAAA GAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGTATCAAGGAATTGGGT TCTCAAATCTTGAAGGAACACCCAGTCGAAAACACCCAATTGCAAAACGAAAA GTTGTACTTGTACTACTTGCAAAACGGTAGAGACATGTACGTCGACCAAGAAT TGGACATCAACAGATTGTCTGACTACGACGTCGACCACATCGTCCCACAATCT TTCTTGAAGGACGACTCTATCGACAACAAGGTCTTGACCAGATCTGACAAGAA CAGAGGTAAGTCTGACAACGTCCCATCTGAAGAAGTCGTCAAGAAGATGAAG AACTACTGGAGACAATTGTTGAACGCCAAGTTGATCACCCAAAGAAAGTTCGA CAACTTGACCAAGGCCGAAAGAGGTGGTTTGTCTGAATTGGACAAGGCCGGT TTCATCAAGAGACAATTGGTCGAAAACCAGACAAATCACCAAGCACGTCGCCCA AATCTTGGACAGCAGAATGAACACCAAGTACGACGAAAACGACAAGTTGATCA GAGAAGTCAAGGTCATCACCTTGAAGTCTAAGTTGGTCTCTGACTTCAGAAAG GACTTCCAATTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCCCACGA CGCCTACTTGAACGCCGTCGTCGGTACCGCCTTGATCAAGAAGTACCCAAAG TTGGAATCTGAATTCGTCTACGGTGACTACAAGGTCTACGACGTCAGAAAGAT
Description Sequence SEQ ID No. GATCGCCAAGTCTGAACAAGAAATCGGTAAGGCCACCGCCAAGTACTTCTTCT ACTCTAACATCATGAACTTCTTCAAGACCGAAATCACCTTGGCCAACGGTGAA ATCAGAAAGAGACCATTGATCGAAACCAACGGTGAAACCGGTGAAATCGTCTG GGACAAGGGTAGAGACTTCGCCACCGTCAGAAAGGTCTTGTCTATGCCACAA GTCAACATCGTCAAGAAGACCGAAGTCCAAACCGGTGGTTTCTCTAAGGAATC TATCTTGCCAAAGAGAAACTCTGACAAGTTGATCGCCAGAAAGAAGGACTGGG ACCCAAAGAAGTACGGTGGTTTCGACTCTCCAACCGTCGCCTACTCTGTCTTG GTCGTCGCCAAGGTCGAAAAGGGTAAGTCTAAGAAGTTGAAGTCTGTCAAGG AATTGTTGGGTATCACCATCATGGAAAGATCTTCTTTCGAAAAGAACCCAATCG ACTTCTTGGAAGCCAAGGGTTACAAGGAAGTCAAGAAGGACTTGATCATCAAG TTGCCAAAGTACTCTTTGTTCGAATTGGAAAACGGTAGAAAGAGAATGTTGGC CTCTGCCGGTGAATTGCAAAAGGGTAACGAATTGGCTTGCCATCTAAGTACG TCAACTTCTTGTACTTGGCCTCTCACTACGAAAAGTTGAAGGGTTCTCCAGAA GACAACGAACAAAAGCAATTGTTCGTCGAACAACACAAGCACTACTTGGACGA AATCATCCGAACAAATCTCTGAATTCTCTAAGAGAGTCATCTTGGCCGACGCCA ACTTGGACAAGGTCTTGTCTGCCTACAACAAGCACAGAGACAAGCCAATCAGA GAAAAAGCCGAAAACATCATCCACTTGTTCACCTTGACCAACTTGGGTGCCCC AGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCTCTA CCAAGGAAGTCTTGGACGCCACTTGATCCACCAATCTATCACCGGTTTGTAC GAAACCAGAATCGACTTGTCTCAATTGGGTGGTGACGGTGGTGGTTCTCCAAA
GAAGAAGAGAAAGGTCTAA ORF cas9 ATGGATAAAAAATATTCTATTGGTTTAGATATTGGTACTAATTCTGTTGGTTGG using codons GCTGTTATTACTGATGAATATAAAGTTCCTTCTAAAAAATTTAAAGTTTTAGGTA U-rich ATACTGATCGTCATTCTATTAAAAAAAATTTAATTGGTGCTTTATTATTTGATTCT Table 4, beginning with GGTGAAACTGCTGAAGCTACTCGTTTAAAACGTACTGCTCGTCGTCGTTATAC and stop codons TCGTCGTAAAAATCGTATTTGTTATTTACAAGAAATTTTTTCTAATGAAATGGCT AAAGTTGATGATTCTTTTTTTCATCGTTTAGAAGAATCTTTTTTAGTTGAAGAAG ATAAAAAAACATGAACGTCATCCTATTTTTGGTAATATTGTTGATGAAGTTGCTTA TCATGAAAAAATATCCTACTATTTATCATTTACGTAAAAAATTAGTTGATTCTACT GATAAAGCTGATTTACGTTTAATTTATTTAGCTTTAGCTCATATGATTAAATTTC GTGGTCATTTTTTAATTGAAGGTGATTTAAATCCTGATAATTCTGATGTTGATAA ATTATTTATTCAATTAGTTCAAACTTATAATCAATTATTTGAAGAAAATCCTATT ATGCTTCTGGTGTTGATGCTAAAGCTATTTTATCTGCTCGTTTATCTAAATCTC GTCGTTTAGAAAATTTTAATTGCTCAATTACCTGGTGAAAAAAAAAATGGTTTATT TGGTAATTTAATTGCTTTATCTTTAGGTTTAACTCCTAATTTTAAATCTAATTTTG
ATTTAGCTGAAGATGCTAAATTACAATTATCTAAAGATACTTATGATGATGATTT AGATAATTTATTAGCTCAAATTGGTGATCAATATGCTGATTTATTTTTAGCTGCT 108
Description Sequence SEQ ID No. AAAAATTTATCTGATGCTATTTTATTATCTGATATTTTACGTGTTAATACTGAAAT TACTAAAGCTCCTTTATCTGCTTCTATGATTAAACGTTATGATGAACATCATCAA GATTTAACTTTATTAAAAGCTTTAGTTCGTCAACAATTACCTGAAAAATATAAAG AAATTTTTTTTGATCAATCTAAAAATGGTTATGCTGGTTATATTGATGGTGGTGC TTCTCAAGAAGAATTTTATAAATTTATTAAACCTATTTTAGAAAAAATGGATGGT ACTGAAGAATTATTAGTTAAATTAAATCGTGAAGATTTATTACGTAAACAACGTA CTTTTGATAATGGTTCTATTCCTCATCAAATTCATTTAGGTGAATTACATGCTAT TTTACGTCGTCAAGAAGATTTTTATCCTTTTTTAAAAGATAATCGTGAAAAAATT GAAAAAATTTTAACTTTTCGTATTCCTTATTATGTTGGTCCTTTAGCTCGTGGTA ATTCTCGTTTTGCTTGGATGACTCGTAAATCTGAAGAAACTATTACTCCTTGGA ATTTTGAAGAAGTTGTTGATAAAGGTGCTTCTGCTCAATCTTTTATTGAACGTA TGACTAATTTTGATAAAAATTTACCTAATGAAAAGTTTACCTAAACATTCTTT ATTATATGAATATTTTACTGTTTATAATGAATTAACTAAAGTTAAATATGTTACTG AAGGTATGCGTAAACCTGCTTTTTTATCTGGTGAACAAAAAAAAGCTATTGTTG ATTTATTATTTAAAACTAATCGTAAAGTTACTGTTAAACAATTAAAAGAAGATTA TTTTAAAAAAATTGAATGTTTTGATTCTGTTGAAATTTCTGGTGTTGAAGATCGT TTTAATGCTTCTTTAGGTACTTATCATGATTTATTAAAAATTATTAAAGATAAAGA TTTTTTAGATAATGAAGAAAATGAAGATATTTTAGAAGATATTGTTTTAACTTTA ACTTTATTTGAAGATCGTGAAATGATTGAAGAACGTTTAAAAACTTATGCTCATT TATTTGATGATAAAGTTATGAAACAATTAAAACGTCGTCGTTATACTGGTTGGG GTCGTTTATCTCGTAAATTAATTAATGGTATTCGTGATAAACAATCTGGTAAAA CTATTTTAGATTTTTTAAAATCTGATGGTTTTGCTAATCGTAATTTTATGCAATTA ATTCATGATGATTCTTTAACTTTTAAAGAAGATATTCAAAAGCTCAAGTTTCTG GTCAAGGTGATTCTTTACATGAACATATTGCTAATTTAGCTGGTTCTCCTGCTA TTAAAAAAGGTATTTTACAAACTGTTAAAGTTGTTGATGAATTAGTTAAAGTTAT GGGTCGTCATAAACCTGAAAATATTGTTATTGAAATGGCTCGTGAAAATCAAAC TACTCAAAAAAGGTCAAAAAAATTCTCGTGAACGTATGAACGTATTGAAGAAG GTATTAAAGAATTAGGTTCTCAAATTTTAAAAGAACATCCTGTTGAAAATACTCA ATTACAAAATGAAAAATTATATTTATATTATTTACAAAATGGTCGTGATATGTAT GTTGATCAAGAATTAGATATTAATCGTTTATCTGATTATGATGTTGATCATATTG TTCCTCAATCTTTTTTAAAAGATGATTCTATTGATAATAAAGTTTTAACTCGTTCT GATAAAAAATCGTGGTAAATCTGATAATGTTCCTTCTGAAGAAGTTGTTAAAAAA ATGAAAAATTATTGGCGTCAATTATTAAATGCTAAATTAATTACTCAACGTAAAT TTGATAATTTAACTAAAGCTGAACGTGGTGGTTTATCTGAATTAGATAAAGCTG GTTTTATTAAACGTCAATTAGTTGAAACTCGTCAAATTACTAAACATGTTGCTCA AATTTTAGATTCTCGTATGAATACTAAATATGATGAAAATGATAAATTAATTCGT GAAGTTAAAGTTATTACTTTAAAATCTAAATTAGTTTCTGATTTTCGTAAAGATT
Description Sequence SEQ ID No. TTCAATTTTATAAAGTTCGTGAAATTAATAATTATCATCATGCTCATGATGCTTA TTTAAATGCTGTTGTTGGTACTGCTTTAATTAAAAAATATCCTAAATTAGAATCT GAATTTGTTTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAAT CTGAACAAAAGAAATTGGTAAAGCTACTGCTAAATATTTTTTTTATTCTAATATTAT GAATTTTTTTAAAACTGAAATTACTTTAGCTAATGGTGAATTCGTAAACGTCCT TTAATTGAAACTAATGGTGAAACTGGTGAAATTGTTTGGGATAAAGGTCGTGAT TTTGCTACTGTTCGTAAAGTTTTATCTATGCCTCAAGTTAATATTGTTAAAAAAAA CTGAAGTTCAAACTGGTGGTTTTTCTAAAGAATCTATTTTACCTAAACGTAATTC TGATAAATTAATTGCTCGTAAAAAAGATTGGGATCCTAAAAAATATGGTGGTTT TGATTCTCCTACTGTTGCTTATTCTGTTTTAGTTGTTGCTAAAGTTGAAAAAAGGT AAATCTAAAAAATTAAAATCTGTTAAAGAATTATTAGGTATTACTATTATGGAAC GTTCTTCTTTTGAAAAAAATCCTATTGATTTTTTAGAAGCTAAAGGTTATAAAGA AGTTAAAAAAGATTTAATTATTAAATTACCTAAATATTCTTTATTTGAATTAGAAA ATGGTCGTAAACGTATGTTAGCTTCTGCTGGTGAATTACAAAAAGGTAATGAAT TAGCTTTACCTTCTAAATATGTTAATTTTTTATATTTAGCTTCTCATTATGAAAA TTAAAAGGTTCTCCCTGAAGATAATGAACAAAAACAATTATTTGTTGAACAACAT AAACATTATTTAGATGAAATTATTGAACAAATTTCTGAATTTTCTAAACGTGTTA TTTTAGCTGATGCTAATTTAGATAAAGTTTTATCTGCTTATAATAAACATCGTGA TAAACCTATTCGTGAACAAGCTGAAAATATTATTCATTTATTTACTTTAACTAAT TTAGGTGCTCCTGCTGCTTTTAAATATTTTGATACTACTATTGATCGTAAACGTT ATACTTCTACTAAAGAAGTTTTAGATGCTACTTTAATTCATCAATCTATTACTGG TTTATATGAAACTCGTATTGATTTATCTCAATTAGGTGGTGATGGTGGTGGTTC
TCCTAAAAAAAAACGTAAAGTTTGA ORF cas9 ATGGACAAAAAATACTCCATCGGCCTCGACATCGGCACCAACTCCGTCGGCT using codons GGGCCGTCATCACCGACGAATACAAAGTCCCCTCCAAAAAATTCAAAGTCCTC G below the GGCAACACCGACAGACACTCCATCAAAAAAAACCTCATCGGCGCCCTCCTCTT Table 4 beginning of stop codons and CGACTCCGGCGAAACCGCCGAAGCCACCAGACTCAAAAGAACCGCCAGAAG AAGATACACCAGAAGAAAAAACAGAATCTGCTACCTCCAAGAAATCTTCTCCA ACGAAATGGCCAAAGTCGACGACTCCTTCTTCCACAGACTCGAAGAATCCTTC CTCGTCGAAGAAGACAAAAAACACGAAAAGACCCCCCATCTTCGGCAACATCGT CGACGAAGTCGCCTACCACGAAAAATACCCCACCATCTACCACCTCAGAAAAA AACTCGTCGACTCCACCGACAAAGCCGACCTCAGACTCATCTACCTCGCCCTC GCCCACATGATCAAATTCAGAGGCCACTTCCTCATCGAAGGCGACCTCAACC CCGACAACTCCGACGTCGACAAACTCTTCATCCAACTCGTCCAAACCTACAAC CAACTCTTCGAAGAAAACCCCATCAACGCCTCCGGCGTCGACGCCAAAGCCA
TCCTCTCCGCCAGACTCTCCAAATCCAGAAGACTCGAAAACCTCATCGCCCAA CTCCCCGGCGAAAAAAAAAAGGCCTCTTCGGCAACCTCATCGCCCTCTCCC 109
Description Sequence SEQ ID No. TCGGCCTCACCCCCAACTTCAAATCCAACTTCGACCCTGCCGAAGACGCCAAA CTCCAACTCTCCAAAGACACCTACGACGACGACCTCGACAACCTCCTCGCCC AAATCGGCGACCAATACGCCGACCTCTTCCTCGCCGCCAAAAACCTCTCCGA CGCCATCCTCCTCTCCGACATCCTCAGAGTCAACACCGAAATCACCAAAGCCC CCCTCTCCGCCTCCATGATCAAAAAGATACGACGAACACCACCAAAGACCTCACC CTCCTCAAAGCCCTCGTCAGACAACAACTCCCCGAAAAATACAAAGAAATCTT CTTCGACCAATCCAAAAACGGCTACGCCGGCTACATCGACGGCGGCGCCTCC CAAGAAGAATTCTACAAATTCATCAAACCCATCCTCGAAAAAATGGACGGCAC CGAAGAACTCCCTCGTCAAACTCAACAGAGAAGACCTCCTCAGAAAACAAAGAA CCTTCGACAACGGCTCCATCCCCCACCAAATCCACCTCGGCGAACTCCACGC CATCCTCAGAAGACAAGAAGACTTCTACCCCTTCCTCAAAGACAACAGAGAAA AAATCGAAAAAATCCTCACCTTCAGAATCCCCTACTACGTCGGCCCCCTCGCC AGAGGCAACTCCAGATTCGCCTGGATGACCAGAAAATCCGAAGAAACCATCA CCCCCTGGAACTTCGAAGAAGTCGTCGACAAAGGCGCCTCCGCCCAATCCTT CATCGAAAGAATGACCAACTTCGACAAAAACCTCCCCAACGAAAAAAGTCCTCC CCAAACACTCCCTCCCTACGAATACTTCACCGTCTACAACGAACTCACCAAA GTCAAATACGTCACCCGAAGGCATGAGAAAACCCGCCTTCCTCTCCGGCGAAC AAAAAAAAGCCCATCGTCGACCTCCTCTTCAAAACCAACAGAAAAGTCACCGTC AAACAACTCAAAGAAGACTACTTCAAAAAAAATCGAATGCTTCGACTCCGTCGA AATCTCCGGCGTCGAAGACAGATTCAACGCCTCCCTCGGCACCTACCACGAC CTCCTCAAAATCATCAAAGACAAAGACTTCCTCGACAACGAAGAAAACGAAGA CATCCTCGAAGACATCGTCCTCACCCTCACCCTCTTCGAAGACAGAGAAATGA TCGAAGAAAGACTCAAAACCTACGCCCACCTCTTCGACGACAAAGTCATGAAA CAACTCAAAAGAAGAAGATACACCGGCTGGGGCAGACTCTCCAGAAAACTCA TCAACGGCATCAGAGACAAACAATCCGGCAAAACCATCCCTGACTTCCTCAAA TCCGACGGCTTCGCCAAACAGAAACTTCATGCAACTCATCCACGACGACTCCCT CACCTTCAAAGAAGACATCCAAAAAGCCCAAGTCTCCGGCCAAGGCGACTCC CTCCACGAACACATCGCCAACCTCGCCGGCTCCCCCGCCATCAAAAAAGGCA TCCTCCAAACCGTCAAAGTCGTCGACGAACTCGTCAAAGTCATGGGCAGACA CAAACCCGAAAACATCGTCATCGAAATGGCCAGAGAAAACCAAAACCACCCAAA AAGGCCAAAAAAACTCCAGAGAAAGAATGAAAAAGAATCGAAGAAGGCATCAAA GAACTCGGCTCCCAAATCCTCAAAGAACACCCCGTCGAAAACACCCAACTCCA AAACGAAAAACTCTACCTCTACTACCTCCAAAACGGCAGAGACATGTACGTCG ACCAAGAACTCGACATCAACAGACTCTCCGACTACGACGTCGACCACATCGTC CCCCAATCCTTCCTCAAAGACGACTCCATCGACAACAAAGTCCTCACCAGATC CGACAAAAAACAGAGGCAAATCCGACAACGTCCCCTCCGAAGAAGTCGTCAAA AAAATGAAAAACTACTGGAGACAACTCCTCAACGCCAAACTCATCACCCAAAG
Description Sequence SEQ ID No. AAAATTCGACAACCTCACCAAAGCCGAAAGAGGCGGCCTCTCCGAACTCGAC AAAGCCGGCTTCATCAAAAAGACAACTCGTCGAAACCAGACAAATCACCAAACA CGTCGCCCAAATCCTCGACTCCAGAATGAACACCAAATACGACGAAAACGACA AACTCATCAGAGAAGTCAAAGTCATCACCCTCAAAATCCAAACTCGTCTCCGAC TTCAGAAAAGACTTCCAATTCTACAAAGTCAGAGAAATCAACAACTACCACCAC GCCCACGACGCCTACCTCAACGCCGTCGTCGGCACCGCCCTCATCAAAAAAT ACCCCAAACTCGAATCCGAATTCGTCTACGGCGACTACAAAGTCTACGACGTC AGAAAAAATGATCGCCAAATCCGAACAAGAAATCGGCAAAGCCACCGCCAAATA CTTCTTCTACTCCAACATCATGAACTTCTTCAAAACCGAAATCACCCTCGCCAA CGGCGAAATCAGAAAAAGACCCCTCATCGAAACCAACGGCGAAAACCGGCGAA ATCGTCTGGGACAAAGGCAGAGACTTCGCCACCGTCAGAAAAGTCCTCTCCA TGCCCCAAGTCAACATCGTCAAAAAACCGAAGTCCAAACCGGCGGCTTCTC CAAAGAATCCATCCTCCCCAAAAGAAACTCCGACAAACTCATCGCCAGAAAAA AAGACTGGGACCCCAAAAAAATACGGCGGCTTCGACTCCCCCACCGTCGCCTA CTCCGTCCTCGTCGTCGCCAAAGTCGAAAAAGGCAAATCCAAAAAACTCAAAT CCGTCAAAGAACTCCCTCGGCATCACCATCATGGAAAGATCCTCCTTCGAAAAA AACCCCATCGACTTCCTCGAAGCCAAAGGCTACAAAGAAGTCAAAAAAAGACCT CATCATCAAACTCCCCAAATACTCCCTCTTCGAACTCGAAAACGGCAGAAAAA GAATGCTCGCCTCCGCCGGCGAACTCCAAAAAGGCAACGAACTCGCCCTCCC CTCCAAATACGTCAACTTCCTCTACCTCGCCTCCCACTACGAAAAACTCAAAG GCTCCCCCGAAGACAACGAACAAAAAACAACTCTTCGTCGAACAACACACAAACAC TACCTCGACGAAATCATCGAACAAATCTCCGAATTCTCCAAAAGAGTCATCCT CGCCGACGCCAACCTCGACAAAGTCCTCTCCGCCTACAACAAAACACAGAGAC AAACCCATCAGAGAACAAGCCGAAAACATCATCCACCTCTTCACCCTCACCAA CCTCGGCGCCCCCGCCGCCTTCAAATACTTCGACACCACCATCGACAGAAAA AGATACACCTCCACCAAAAGAAGTCCTCGACGCCACCCTCATCCACCAATCCAT CACCGGCCTCTACGAAAACCAGAATCGACCTCTCCCAACTCGGCGGCGACGGC
GGCGGCTCCCCCAAAAAAAAAAGAAAAGTCTGA ORF cas9 ATGGATAAGAAGTATAGTATTGGATTGGATATTGGAACAAATAGTGTGGGATG using codons GGCTGTGATTACAGATGAGTATAAGGTGCCTAGTAAGAAGTTTAAGGTGTTGG C below the GAAATACAGATAGACATAGTATTAAGAAGAATTTGATTGGAGCTTTGTTGTTTG Table 4, beginning with ATAGTGGAGAGACAGCTGAGGCTACAAGATTGAAGAGAACAGCTAGAAGAAG and stop codons ATATACAAGAAGAAAGAATAGAATTTGTTATTTGCAGGAGATTTTTAGTAATGA GATGGCTAAGGTGGATGATAGTTTTTTTCATAGATTGGAGGAGAGTTTTTTGGT GGAGGAGGATAAGAAGCATGAGAGACATCCTATTTTTGGAAATATTGTGGATG
AGGTGGCTTATCATGAGAAGTATCCTACAATTTATCATTTGAGAAAGAAGTTGG TGGATAGTACAGATAAGGCTGATTTGAGATTGATTTATTTGGCTTTGGCTCATA 110
Description Sequence SEQ ID No. TGATTAAGTTTAGAGGACATTTTTTGATTGAGGGAGATTTGAATCCTGATAATA GTGATGTGGATAAGTTGTTTATTCAGTTGGTGCAGACATATAATCAGTTGTTTG AGGAGAATCCTATTAATGCTAGTGGAGTGGATGCTAAGGCTATTTTGAGTGCT AGATTGAGTAAGAGTAGAAGATTGGAGAATTTGATTGCTCAGTTGCCTGGAGA GAAGAAGAATGGATTGTTTGGAAATTTGATTGCTTTGAGTTTGGGATTGACAC CTAATTTTAAGAGTAATTTTGATTTGGCTGAGGATGCTAAGTTGCAGTTGAGTA AGGATACATATGATGATGATTTGGATAATTTGTTGGCTCAGATTGGAGATCAGT ATGCTGATTTGTTTTTGGCTGCTAAGAATTTGAGTGATGCTATTTTGTTGAGTG ATATTTTGAGAGTGAATACAGAGATTACAAAGGCTCCTTTGAGTGCTAGTATGA TTAAGAGATATGATGAGCATCATCAGGATTTGACATTGTTGAAGGCTTTGGTG AGACAGCAGTTGCCTGAGAAGTATAAGGAGATTTTTTTTGATCAGAGTAAGAA TGGATATGCTGGATATATTGATGGAGGAGCTAGTCAGGAGGAGTTTTATAAGT TTATTAAGCCTATTTTGGAGAAGATGGATGGAACAGAGGAGTTGTTGGTGAAG TTGAATAGAGAGGATTTGTTGAGAAAGCAGAGAACATTTGATAATGGAAGTATT CCTCATCAGATTCATTTGGGAGAGTTGCATGCTATTTTGAGAAGACAGGAGGA TTTTTATCCTTTTTTGAAGGATAATAGAGAGAAGATTGAGAAGATTTTGACATTT AGAATTCCTTATTATTGTGGGACCTTTGGCTAGAGGAAATAGTAGATTTGCTTG GATGACAAGAAAGAGTGAGGAGACAATTACACCTTGGAATTTTGAGGAGGTG GTGGATAAGGGAGCTAGTGCTCAGAGTTTTATTGAGAGAATGACAAATTTTGA TAAGAATTTGCCTAATGAGAAGGTGTTGCCTAAGCATAGTTTGTTGTATGAGTA TTTTACAGTGTATAATGAGTTGACAAAGGTGAAGTATGTGACAGAGGGAATGA GAAAGCCTGCTTTTTTGAGTGGAGAGCAGAAGAAGGCTATTGTGGATTTGTTG TTTAAGACAAATAGAAAGGTGACAGTGAAGCAGTTGAAGGAGGATTATTTTAA GAAGATTGAGTGTTTTGATAGTGTGGAGATTAGTGGAGTGGAGGATAGATTTA ATGCTAGTTTGGGAACATATCATGATTTGTTGAAGATTATTAAGGATAAGGATT TTTTGGATAATGAGGAGAATGAGGATATTTTGGAGGATATTGTGTTGACATTGA CATTGTTTGAGGATAGAGAGATGATTGAGGAGAGATTGAAGACATATGCTCAT TTGTTTGATGATAAGGTGATGAAGCAGTTGAAGAGAAGAAGATATACAGGATG GGGAAGATTGAGTAGAAAGTTGATTAATGGAATTAGAGATAAGCAGAGTGGAA AGACAATTTTGGATTTTTTGAAGAGTGATGGATTTGCTAATAGAAATTTTATGC AGTTGATTCATGATGATAGTTTGACATTTAAGGAGGATATTCAGAAGGCTCAG GTGAGTGGACAGGGAGATAGTTTGCATGAGCATATTGCTAATTTGGCTGGAAG TCCTGCTATTAAGAAGGGAATTTTGCAGACAGTGAAGGTGGTGGATGAGTTGG TGAAGGTGATGGGAAGACATAAGCCTGAGAATATTGTGATTGAGATGGCTAGA GAGAATCAGACAACACAGAAGGGACAGAAGAATAGTAGAGAGAGAATGAAGA GAATTGAGGAGGGAATTAAGGAGTTGGGAAGTCAGATTTTGAAGGAGCATCC TGTGGAGAATACACAGTTGCAGAATGAGAAGTTGTATTTGTATTATTTGCAGAA
Description Sequence SEQ ID No. TGGAAGAGATATGTATGTGGATCAGGAGTTGGATATTAATAGATTGAGTGATT ATGATGTGGATCATATTGTGCCTCAGAGTTTTTTGAAGGATGATAGTATTGATA ATAAGGTGTTGACAAGAAGTGATAAGAATAGAGGAAAGAGTGATAATGTGCCT AGTGAGGAGGTGGTGAAGAAGATGAAGAATTATTGGAGACAGTTGTTGAATG CTAAGTTGATTACACAGAGAAAGTTTGATAATTTGACAAAGGCTGAGAGAGGA GGATTGAGTGAGTTGGATAAGGCTGGATTTATTAAGAGACAGTTGGTGGAGAC AAGACAGATTACAAAGCATGTGGCTCAGATTTTGGATAGTAGAATGAATACAA AGTATGATGAGAATGATAAGTTGATTAGAGAGGTGAAGGTGATTACATTGAAG AGTAAGTTGGTGAGTGATTTTAGAAAGGATTTTCAGTTTTATAAGGTGAGAGAG ATTAATAATTATCATCATGCTCATGATGCTTATTTGAATGCTGTGGTGGGAACA GCTTTGATTAAGAAGTATCCTAAGTTGGAGAGTGAGTTTGTGTATGGAGATTAT AAGGTGTATGATGTGAGAAAGATGATTGCTAAGAGTGAGGCAGGAGATTGGAAA GGCTACAGCTAAGTATTTTTTTTATAGTAATATTATGAATTTTTTTAAAGACAGAG ATTACATTGGCTAATGGAGAGATTAGAAAGAGACCTTTGATTGAGACAAATGG AGAGACAGGAGAGATTGTGTGGGATAAGGGAAGAGATTTTGCTACAGTGAGA AAGGTGTTGAGTATGCCTCAGGTGAATATTGTGAAGAAGACAGAGGTGCAGA CAGGAGGATTTAGTAAGGAGAGTATTTTGCCTAAGAGAAATAGTGATAAGTTG ATTGCTAGAAAGAAGGATTGGGATCCTAAGAAGTATGGAGGATTTGATAGTCC TACAGTGGCTTATAGTGTGTTGGTGGTGGCTAAGGTGGAGAAGGGAAAGAGT AAGAAGTTGAAGAGTGTGAAGGAGTTGTTGGGAATTACAATTATGGAGAGAAG TAGTTTTGAGAAGAATCCTATTGATTTTTTGGAGGCTAAGGGATATAAGGAGGT GAAGAAGGATTTGATTATTAAGTTGCCTAAGTATAGTTTGTTTGAGTTGGAGAA TGGAAGAAAGAGAATGTTGGCTAGTGCTGGAGAGTTGCAGAAGGGAAATGAG TTGGCTTTGCCTAGTAAGTATGTGAATTTTTTGTATTTGGCTAGTCATTATGAG AAGTTGAAGGGAAGTCCTGAGGATAATGAGCAGAAGCAGTTGTTTGTGGAGC AGCATAAGCATTATTTGGATGAGATTATTGAGCAGATTAGTGAGTTTAGTAAGA GAGTGATTTTGGCTGATGCTAATTTGGATAAGGTGTTGAGTGCTTATAATAAGC ATAGAGATAAGCCTATTAGAGAGCAGGCTGAGAATATTATTCATTTGTTTACAT TGACAAATTTGGGAGCTCCTGCTGCTTTTAAGTATTTTGATACAACAATTGATA GAAAGAGATATACAAGTACAAAGGAGGTGTTGGATGCTACATTGATTCATCAG AGTATTACAGGATTGTATGAGACAAGAATTGATTTGAGTCAGTTGGGAGGAGA
TGGAGGAGGAAGTCCTAAGAAGAAGAGAAAGGTGTGA ORF cas9 ATGGACAAGAAGTACTCCATCGGCCTGGACATCGGCACCAACTCCGTGGGCT GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT using codons GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT The bottom of Table 4 with GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT start codons 111
Description Sequence SEQ ID No. and stop TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA
Description Sequence SEQ ID No. GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCG TGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCG GTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACC CAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAG CTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATC ACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACG AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCT GGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAAC AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCC CTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCA AGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACC GAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACC AACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACC GTGCGGAAGGTGCTGTCCATGCCCAGGTGAACATCGTGAAGAAGACCGAG GTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCC GACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGC TTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGA AGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCAT CATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAG GGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCC TGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGC TGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTA CCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCA GAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAG CAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACA AGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGG CCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGC CTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAG GAGGTGCTGGACGCCACCCCTGATCCACCAGTCCATCACCGGCCTGTACGAGA
Description Sequence SEQ ID No. CCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCAAGA
AGAAGCGGAAGGTGTGA ORF cas9 ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT A / U low GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4 with GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC start codons and stop GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA
GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA 112
Description Sequence SEQ ID No. TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT
Description Sequence SEQ ID No. TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGC
AGCCCCAAGAAGAAGCGGAAGGTGTGA ORF cas9 ATGGACAAGAAGTACTCCATCGGCCTGGACATCGGCACCAACTCCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT A low GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4 with two sequences GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT terminal NLS TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA C and GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG codons start and stop CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC
GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG 113
Description Sequence SEQ ID No. TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCG TGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCG GTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACC CAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAG CTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATC ACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACG AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCT GGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAAC AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCC CTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCA AGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACC GAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACC AACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACC GTGCGGAAGGTGCTGTCCATGCCCAGGTGAACATCGTGAAGAAGACCGAG GTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCC
Description Sequence SEQ ID No. GACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGC TTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGA AGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCAT CATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAG GGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCC TGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGC TGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTA CCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCA GAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAG CAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACA AGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGG CCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGC CTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAG GAGGTGCTGGACGCCACCCCTGATCCACCAGTCCATCACCGGCCTGTACGAGA CCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCAAGA AGAAGCGGAAGGTGGACGGCTCCCCCAAGAAGAAGCGGAAGGTGGACTCCG
GCTGA OR9 cas9 of ATGGACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCT nickase using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT The GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC below Table 4, beginning with the codons GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA and stop GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC
ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGC 114
Description Sequence SEQ ID No. CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCG TGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCG GTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACC CAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAG CTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATC ACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACG AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCT GGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAAC
Description Sequence SEQ ID No. AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCC CTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCA AGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACC GAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACC AACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACC GTGCGGAAGGTGCTGTCCATGCCCAGGTGAACATCGTGAAGAAGACCGAG GTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCC GACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGC TTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGA AGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCAT CATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAG GGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCC TGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGC TGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTA CCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCA GAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAG CAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACA AGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGG CCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGC CTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAG GAGGTGCTGGACGCCACCCCTGATCCACCAGTCCATCACCGGCCTGTACGAGA CCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCAAGA
AGAAGCGGAAGGTGTGA OR9 cas9 of ATGGACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCT nickase using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT The GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC below Table 4, beginning with the codons GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA and stop without GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG NLS CAACATCGTGGACGAGGTGGCCTACACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT
GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG 115
Description Sequence SEQ ID No. AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCG TGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCG
Description Sequence SEQ ID No. GTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACC CAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAG CTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATC ACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACG AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCT GGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAAC AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCC CTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCA AGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACC GAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACC AACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACC GTGCGGAAGGTGCTGTCCATGCCCAGGTGAACATCGTGAAGAAGACCGAG GTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCC GACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGC TTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGA AGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCAT CATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAG GGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCC TGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGC TGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTA CCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCA GAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAG CAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACA AGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGG CCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGC CTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAG GAGGTGCTGGACGCCACCCCTGATCCACCAGTCCATCACCGGCCTGTACGAGA
CCCGGATCGACCTGTCCCAGCTGGGCGGCGACTGA ORF cas9 ATGGACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCT nickase using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT The GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC below Table 4, two GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA NLS sequences and C-terminal GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG 116
Description Sequence SEQ ID No. start codons CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC and stop CTGCGGAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC
Description Sequence SEQ ID No. CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCG TGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCG GTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGT GAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACC CAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAG CTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATC ACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACG AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCT GGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAAC AACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCC CTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCA AGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACC GAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACC AACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACC GTGCGGAAGGTGCTGTCCATGCCCAGGTGAACATCGTGAAGAAGACCGAG GTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCC GACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGC TTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGA AGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCAT CATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAG GGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCC TGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGC TGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTA CCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCA GAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAG CAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACA AGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGG CCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGC CTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAG GAGGTGCTGGACGCCACCCCTGATCCACCAGTCCATCACCGGCCTGTACGAGA CCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCAAGA AGAAGCGGAAGGTGGACGGCTCCCCCAAGAAGAAGCGGAAGGTGGACTCCG
Description Sequence SEQ ID No.
GCTGA ORF dCas9 ATGGACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCT using GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT The codons of GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT Table 4 below, with start and stop codons GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA
CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAAGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA 117
Description Sequence SEQ ID No. AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACGCCATC GTGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCC GGTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGG TGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCAC CCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGA GCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGAT CACCAAGCACGTGGCCCAGATCCTGGACTCCCGGAATGAACACCAAGTACGAC GAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGC TGGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAA CAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGC CCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTAC AAGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGC AAGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGAC CGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGAC CAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCAC CGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGA GGTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTC CGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGG CTTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGA GAAGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCAC CATCATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCC AAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACT CCCTGTTCGAGCTGGAGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCG AGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCT
Description Sequence SEQ ID No. GTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGA GCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGG ACAAGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGC AGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGC CGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACC AAGGAGGTGCTGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACG AGACCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCA
AGAAGAAGCGGAAGGTGTGA ORF dCas9 ATGGACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT A low GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4, beginning with the codons and stop GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT without TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA NLS GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC
GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG 118
Description Sequence SEQ ID No. TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACGCCATC GTGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCC GGTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGG TGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCAC CCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGA GCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGAT CACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGAC GAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGC TGGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAA CAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGC CCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTAC AAGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGC AAGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGAC CGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGAC CAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCAC CGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGA GGTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTC
Description Sequence SEQ ID No. CGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGG CTTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGA GAAGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCAC CATCATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCC AAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACT CCCTGTTCGAGCTGGAGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCG AGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCT GTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGA GCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGG ACAAGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGC AGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGC CGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACC AAGGAGGTGCTGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACG
AGACCCGGATCGACCTGTCCCAGCTGGGCGGCGACTGA ORF dCas9 ATGGACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCT A low GGGCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4 with two GTTCGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT NLS sequences C-terminal codons and TCTCCAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGA GTCCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG start and stop CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC CTGCGGAAGAAGCTGGTGGACTCCACGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTG GACGCCAAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAG AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAAC CTGATCGCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCT GGCCGAGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCT GGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGC CGCCAAGAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAAC ACCGAGATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC
CCGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCC 119
Description Sequence SEQ ID No. ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCT GGATGACCCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTT CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTAC GAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGG GCATGCGGAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGG ACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGA CTACTTCAAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAG GACCGGTTCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCA AGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT CGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGG CGGCGGTACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATC CGGGACAAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCT TCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAA GGAGGACATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGA GCACATCGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCA GACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGG CCAGAAGAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGA GCTGGGCTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTG GACCAGGAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACGCCATC GTGCCCCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCC GGTCCGACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGG TGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCAC CCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGA GCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGAT CACCAAGCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGAC GAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGC TGGTGTCCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAA CAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGC CCTGATCAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTAC
Description Sequence SEQ ID No. AAGGTGTACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGC AAGGCCACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGAC CGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGAC CAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCAC CGTGCGGAAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGA GGTGCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTC CGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGG CTTCGACTCCCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGA GAAGGGCAAGTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCAC CATCATGGAGCGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCC AAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACT CCCTGTTCGAGCTGGAGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCG AGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCT GTACCTGGCCTCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGA GCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCTCCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGG ACAAGGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGC AGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGC CGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACC AAGGAGGTGCTGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACG AGACCCGGATCGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCA AGAAGAAGCGGAAGGTGGACGGCTCCCCCAAGAAGAAGCGGAAGGTGGACT
CCGGCTGA ORF cas9 ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT A / U low GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4 with two GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT NLS sequences C-terminal codons and TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG start and stop CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG
GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA 120
Description Sequence SEQ ID No. CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG
Description Sequence SEQ ID No. GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCA GCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGG
TGGACAGCGGCTGA ORF cas9 ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT A / U low GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT in Table 4, with codons GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT start and stop 121 and TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA
Description Sequence SEQ ID No. without NLS GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC
Description Sequence SEQ ID No. TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACTGA
Description Sequence SEQ ID No. ORF cas9 ATGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCT nickase GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT codons using A / U GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC below Table 4, beginning with the codons GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA and stop GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA
TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG 122
Description Sequence SEQ ID No. GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT
Description Sequence SEQ ID No. CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGC
AGCCCCAAGAAGAAGCGGAAGGTGTGA ORF 9 Cas ATGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCT nickase GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT codons using A / U GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC below Table 4, two GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA NLS sequences and C-terminal GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG start codons and stop CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC CTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA
GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG 123
Description Sequence SEQ ID No. TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT
Description Sequence SEQ ID No. GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCA GCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGG
TGGACAGCGGCTGA ORF cas9 ATGGACAAGAAGTACAGCATCGGCCTGGcCATCGGCACCAACAGCGTGGGCT nickase GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT codons using A / U GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC below Table 4 beginning of GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA stop codons and without GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG NLS CAACATCGTGGACGAGGTGGCCTACACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT
GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC 124
Description Sequence SEQ ID No. CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA
Description Sequence SEQ ID No. CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC
TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACTGA ORF dCas9 ATGGACAAGAAGTACAGCATCGGCCTGGcCATCGGCACCAACAGCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT A / U low GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4 with GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC start codons and stop GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA
CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC 125
Description Sequence SEQ ID No. CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC
TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACgcC ATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGA CCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA
Description Sequence SEQ ID No. GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGC
AGCCCCAAGAAGAAGCGGAAGGTGTGA ORF dCas9 ATGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCT GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT codons using A / U low GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4 with two GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT NLS sequences C-terminal codons and TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG start and stop CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCAC 126 CTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATC
Description Sequence SEQ ID No. TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA
Description Sequence SEQ ID No. AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACGC CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCA GCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGG TGGACAGCGGCTGA
Description Sequence SEQ ID No. ORF dCas9 ATGGACAAGAAGTACAGCATCGGCCTGGcCATCGGCACCAACAGCGTGGGCT using codons GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCT A / U low GGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCT of Table 4, beginning with GTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCC GGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCT and stop codons and without TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA NLS GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGG CAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACACAC CTGCGGAAGAAGCTGGTGGACAGCCACCGACAAGGCCGACCTGCGGCTGATC TACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT GCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTG GACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGA GAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAA CCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGAC CTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGAC CTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGA ACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGA CGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCT GCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGC CCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACC GGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC ACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACT TCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTT CCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGC CTGGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGA GGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG TACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCG TGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTG GAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCA
TCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG 127
Description Sequence SEQ ID No. GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC
TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACgcC ATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGA CCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT
Description Sequence SEQ ID No. CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC
TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACTGA ORF Nme ATGGCCGCCTTCAAGCCCAACTCCATCAACTACATCCTGGGCCTGGACATCG cas9 using codons GCATCGCCTCCGTGGGCTGGGCCATGGTGGAGATCGACGAGGAGGAGAACC The CCATCCGGCTGATCGACCTGGGCGTGCGGGTGTTCGAGCGGGCCGAGGTGC CCAAGACCGGCGACTCCCTGGCCATGGCCCGGCGGCTGGCCCGGTCCGTGC below Table 4, beginning with the codons GGCGGCTGACCCGGCGGCGGGCCCACCGGCTGCTGCGGACCCGGCGGCTG CTGAAGCGGGAGGGCGTGCTGCAGGCCGCCAACTTCGACGAGAACGGCCTG and stop ATCAAGTCCCTGCCCAACACCCCCTGGCAGCTGCGGGCCGCCGCCCTGGAC CGGAAGCTGACCCCCCTGGAGTGGTCCGCCGTGCTGCTGCACCTGATCAAG CACCGGGGCTACCTGTCCCAGCGGAAGAACGAGGGCGAGACCGCCGACAAG GAGCTGGGCGCCCTGCTGAAGGGCGTGGCCGGCAACGCCCACGCCCTGCA GACCGGCGACTTCCGGACCCCCGCCGAGCTGGCCCTGAACAAGTTCGAGAA GGAGTCCGGCCACATCCGGAACCAGCGGTCCGACTACTCCCACACCTTCTCC CGGAAGGACCCTGCAGGCCGAGCTGATCCTGCTGTTCGAGAAGCAGAAGGAG TTCGGCAACCCCCACGTGTCCGGCGGCCTGAAGGAGGGCATCGAGACCCTG CTGATGACCCAGCGGCCCGCCCTGTCCGGCGACGCCGTGCAGAAGATGCTG GGCCACTGCACCTTCGAGCCCGCCGAGCCCAAGGCCGCCAAGAACACCTAC ACCGCCGAGCGGTTCATCTGGCTGACCAAGCTGAACAACCTGCGGATCCTGG AGCAGGGCTCCGAGCGGCCCCTGACCGACACCGAGCGGGCCACCCTGATGG ACGAGCCCTACCGGAAGTCCAAGCTGACCTACGCCCAGGCCCGGAAGCTGC TGGGCCTGGAGGACACCGCCTTCTTCAAGGGCCTGCGGTACGGCAAGGACA ACGCCGAGGCCTCCACCCTGATGGAGATGAAGGCCTACCACGCCATCTCCCG GGCCCTGGAGAAGGAGGGCCTGAAGGACAAGAAGTCCCCCCTGAACCTGTC CCCCGAGCTGCAGGACGAGATCGGCACCGCCTTCTCCCTGTTCAAGACCGAC GAGGACATCACCGGCCGGCTGAAGGACCGGATCCAGCCCGAGATCCTGGAG GCCCTGCTGAAGCACATCTCCTTCGACAAGTTCGTGCAGATCTCCCCTGAAGG CCCTGCGGCGGATCGTGCCCCTGATGGAGCAGGGCAAGCGGTACGACGAGG CCTGCGCCGAGATCTACGGCGACCACTACGGCAAGAAGAACACCGAGGAGA AGATCTACCTGCCCCCCATCCCCGCCGACGAGATCCGGAACCCCGTGGTGCT GCGGGCCCTGTCCCAGGCCCGGAAGGTGATCAACGGCGTGGTGCGGCGGTA
CGGCTCCCCCGCCCGGATCCACATCGAGACCGCCCGGGAGGTGGGCAAGTC CTTCAAGGACCGGAAGGAGATCGAGAAGCGGCAGGAGGAGAACCGGAAGGA 128
Description Sequence SEQ ID No. CCGGGAGAAGGCCGCCGCCAAGTTCCGGGAGTACTTCCCCAACTTCGTGGG CGAGCCCAAGTCCAAGGACATCCTGAAGCTGCGGCTGTACGAGCAGCAGCA CGGCAAGTGCCTGTACTCCGGCAAGGAGATCAACCTGGGCCGGCTGAACGA GAAGGGCTACGTGGAGATCGACCACGCCCTGCCCTTCTCCCGGACCTGGGA CGACTCCTTCAACAACAAGGTGCTGGTGCTGGGCTCCGAGAACCAGAACAAG GGCAACCAGACCCCCTACGAGTACTTCAACGGCAAGGACAACTCCCGGGAGT GGCAGGAGTTCAAGGCCCGGGTGGAGACCCTCCCGGTTCCCCCGGTCCAAGA AGCAGCGGATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGCGGA ACCTGAACGACACCCGGTACGTGAACCGGTTCCTGTGCCAGTTCGTGGCCGA CCGGATGCGGCTGACCGGCAAGGGCAAGAAGCGGGTGTTCGCCTCCAACGG CCAGATCACCAACCTGCTGCGGGGCTTCTGGGGCCTGCGGAAGGTGCGGGC CGAGAACGACCGGCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCTCCAC CGTGGCCATGCAGCAGAAGATCACCCGGTTCGTGCGGTACAAGGAGATGAAC GCCTTCGACGGCAAGACCATCGACAAGGAGACCGGCGAGGTGCTGCACCAG AAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGATGATCC GGGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCCGACACCC TGGAGAAGCTGCGGACCCTGCTGGCCGAGAAGCTGTCCTCCCGGCCCGAGG CCGTGCACGAGTACGTGACCCCCTGTTCGTGTCCCGGGCCCCCAACCGGA AGATGTCCGGCCAGGGCCACATGGAGACCGTGAAGTCCGCCAAGCGGCTGG ACGAGGGCGTGTCCGTGCTGCGGGTGCCCCTGACCCAGCTGAAGCTGAAGG ACCTGGAGAAGATGGTGAACCGGGAGCGGGAGCCCAAGCTGTACGAGGCCC TGAAGGCCCGGCTGGAGGCCCACAAGGACGACCCCGCCAAGGCCTTCGCCG AGCCCTTCTACAAGTACGACAAGGCCGGCAACCGGACCCAGCAGGTGAAGG CCGTGCGGGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGTGCGGAACCACA ACGGCATCGCCGACAACGCCACCATGGTGCGGGTGGACGTGTTCGAGAAGG GCGACAAGTACTACCTGGTGCCCATCTACTCCTGGCAGGTGGCCAAGGGCAT CCTGCCCGACCGGGCCGTGGTGCAGGGCAAGGACGAGGAGGACTGGCAGC TGATCGACGACTCCTTCAACTTCAAGTTCTCCCTGCACCCCAACGACCTGGTG GAGGTGATCACCAAGAAGGCCCGGATGTTCGGCTACTTCGCCTCCTGCCACC GGGGCACCGGCAACATCAACATCCGGATCCACGACCTGGACCACAAGATCG GCAAGAACGGCATCCTGGAGGGCATCGGCGTGAAGACCGCCCTGTCCTTCC AGAAGTACCAGATCGACGAGCTGGGCAAGGAGATCCGGCCCTGCCGGCTGA AGAAGCGGCCCCCCGTGCGGTCCGGCAAGCGGACCGCCGACGGCTCCGAG TTCGAGTCCCCCAAAGAAGAAGCGGAAGGTGGAGTGA
Description Sequence SEQ ID No. ORF Nme ATGGCCGCCTTCAAGCCCAACAGCATCAACTACATCCTGGGCCTGGACATCG cas9 GCATCGCCAGCGTGGGCTGGGCCATGGTGGAGATCGACGAGGAGGAGAACC codons using A / U CCATCCGGCTGATCGACCTGGGCGTGCGGGTGTTCGAGCGGGCCGAGGTGC CCAAGACCGGCGACAGCCTGGCCATGGCCCGGCGGCTGGCCCGGAGCGTG below Table 4, beginning with the codons CGGCGGCTGACCCGGCGGCGGGCCCACCGGCTGCTGCGGACCCGGCGGCT GCTGAAGCGGGAGGGCGTGCTGCAGGCCGCCAACTTCGACGAGAACGGCCT and stop GATCAAGAGCCTGCCCAACACCCCCTGGCAGCTGCGGGCCGCCGCCCTGGA CCGGAAGCTGACCCCCCTGGAGTGGAGCGCCGTGCTGCTGCACCTGATCAA GCACCGGGGCTACCTGAGCCAGCGGAAGAACGAGGGCGAGACCGCCGACAA GGAGCTGGGCGCCCTGCTGAAGGGCGTGGCCGGCAACGCCCACGCCCTGC AGACCGGCGACTTCCGGACCCCCGCCGAGCTGGCCCTGAACAAGTTCGAGA AGGAGAGCGGCCACATCCGGAACCAGCGGAGCGACTACAGCCACACCTTCA GCCGGAAGGACCCTGCAGGCCGAGCTGATCCTGCTGTTCGAGAAGCAGAAGG AGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGGGCATCGAGACCC TGCTGATGACCCAGCGGCCCGCCCTGAGCGGCGACGCCGTGCAGAAGATGC TGGGCCACTGCACCTTCGAGCCCGCCGAGCCCAAGGCCGCCAAGAACACCT ACACCGCCGAGCGGTTCATCTGGCTGACCAAGCTGAACAACCTGCGGATCCT GGAGCAGGGCAGCGAGCGGCCCCTGACCGACACCGAGCGGGCCACCCTGA TGGACGAGCCCTACCGGAAGAGCAAGCTGACCTACGCCCAGGCCCGGAAGC TGCTGGGCCTGGAGGACACCGCCTTCTTCAAGGGCCTGCGGTACGGCAAGG ACAACGCCGAGGCCAGCACCCTGATGGAGATGAAGGCCTACCACGCCATCA GCCGGGCCCTGGAGAAGGAGGGCCTGAAGGACAAGAAGAGCCCCCTGAACC TGAGCCCCGAGCTGCAGGACGAGATCGGCACCGCCTTCAGCCTGTTCAAGAC CGACGAGGACATCACCGGCCGGCTGAAGGACCGGATCCAGCCCGAGATCCT GGAGGCCCTGCTGAAGCACATCAGCTTCGACAAGTTCGTGCAGATCAGCCTG AAGGCCCTGCGGCGGATCGTGCCCCTGATGGAGCAGGGCAAGCGGTACGAC GAGGCCTGCGCCGAGATCTACGGCGACCACTACGGCAAGAAGAACACCGAG GAGAAGATCTACCTGCCCCCCATCCCCGCCGACGAGATCCGGAACCCCGTG GTGCTGCGGGCCCTGAGCCAGGCCCGGAAGGTGATCAACGGCGTGGTGCG GCGGTACGGCAGCCCCGCCCGGATCCACATCGAGACCGCCCGGGAGGTGG GCAAGAGCTTCAAGGACCGGAAGGAGATCGAGAAGCGGCAGGAGGAGAACC GGAAGGACCGGGAGAAGGCCGCCGCCAAGTTCCGGGAGTACTTCCCCAACT TCGTGGGCGAGCCCAAGAGCAAGGACATCCTGAAGCTGCGGCTGTACGAGC AGCAGCACGGCAAGTGCCTGTACAGCGGCAAGGAGATCAACCTGGGCCGGC TGAACGAGAAGGGCTACGTGGAGATCGACCACGCCCTGCCCTTCAGCCGGA
CCTGGGACGACAGCTTCAACAACAAGGTGCTGGTGCTGGGCAGCGAGAACC AGAACAAGGGCAACCAGACCCCCTACGAGTACTTCAACGGCAAGGACAACAG 129
Description Sequence SEQ ID No. CCGGGAGTGGCAGGAGTTCAAGGCCCGGGTGGAGACCAGCCGGTTCCCCCG GAGCAAGAAGCAGCGGATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAA GGAGCGGAACCTGAACGACACCCGGTACGTGAACCGGTTCCTGTGCCAGTTC GTGGCCGACCGGATGCGGCTGACCGGCAAGGGCAAGAAGCGGGTGTTCGCC AGCAACGGCCAGATCACCAACCTGCTGCGGGGCTTCTGGGGCCTGCGGAAG GTGCGGGCCGAGAACGACCGGCACCACGCCCTGGACGCCGTGGTGGTGGC CTGCAGCACCGTGGCCATGCAGCAGAAGATCACCCGGTTCGTGCGGTACAAG GAGATGAACGCCTTCGACGGCAAGACCATCGACAAGGAGACCGGCGAGGTG CTGCACCAGAAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGG TGATGATCCGGGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGG CCGACACCCTGGAGAAGCTGCGGACCCTGCTGGCCGAGAAGCTGAGCAGCC GGCCCGAGGCCGTGCACGAGTACGTGACCCCCTGTTCGTGAGGCCGGGCCC CCAACCGGAAGATGAGCGGCCAGGGCCACATGGAGACCGTGAAGAGCGCCA AGCGGCTGGACGAGGGCGTGAGCGTGCTGCGGGTGCCCCTGACCCAGCTGA AGCTGAAGGACCTGGAGAAGATGGTGAACCGGGAGCGGGAGCCCAAGCTGT ACGAGGCCCTGAAGGCCCGGCTGGAGGCCCACAAGGACGACCCCGCCAAG GCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGGACCCAG CAGGTGAAGGCCGTGCGGGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGT GCGGAACCACAACGGCATCGCCGACAACGCCACCATGGTGCGGGTGGACGT GTTCGAGAAGGGCGACAAGTACTACCTGGTGCCCATCTACAGCTGGCAGGTG GCCAAGGGCATCCTGCCCGACCGGGCCGTGGTGCAGGGCAAGGACGAGGA GGACTGGCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGCCTGCACCCC AACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGGATGTTCGGCTACTTCG CCAGCTGCCCACCGGGGCACCGGCAACATCAACATCCGGATCCACGACCTGG ACCACAAGATCGGCAAGAACGGCATCCTGGAGGGCATCGGCGTGAAGACCG CCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGGCAAGGAGATCCGGC CCTGCCGGCTGAAGAAGCGGCCCCCCGTGCGGAGCGGCAAGCGGACCGCC
GACGGCAGCGAGTTCGAGAGCCCCAAGAAGAAGCGGAAGGTGGAGTGA open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS1 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT
CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG 130
Description Sequence SEQ ID No. CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC
Description Sequence SEQ ID No. CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA GCCTGGCAGCAAAGAGAAGCAGAACAACATAG
Description Sequence SEQ ID NO open reading frame of ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS2 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA start and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC
AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC 131
Description Sequence SEQ ID No. GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC
Description Sequence SEQ ID No. AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCCAGGCAGCAAAGAGAAGCAGAACAACATAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS3 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA
ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT 132
Description Sequence SEQ ID No. TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC
Description Sequence SEQ ID No. CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCCCGGCACCGGCAAAGAGAGAAAGAACAACATAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS4 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG
AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA 133
Description Sequence SEQ ID No. GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA
Description Sequence SEQ ID No. AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCCAGGCAGCAAAGAGACCGAGAACAACATAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS5 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC
CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCA 134
Description Sequence SEQ ID No. GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT
Description Sequence SEQ ID No. ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCAGAGCAGCAAAGAGACCGAGAACAACATAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS6 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA
TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT 135
Description Sequence SEQ ID No. CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA
Description Sequence SEQ ID No. ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCGCAGCAGCAAAGAGAAGCTGGAGCATGGCAGCATAG open reading frame for ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS7 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA start and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT 136
Description Sequence SEQ ID No. CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA
Description Sequence SEQ ID No. GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA GCGCAGCAGCAAAGAGAGTCTGGAGCATGGCATTCTAG
Description Sequence SEQ ID NO open reading frame of ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS8 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA start and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC
AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC 137
Description Sequence SEQ ID No. GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC
Description Sequence SEQ ID No. AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCGCAGCAGCAAAGAGAAGCTGGAGCATGGCATTCTAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS9 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA
ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT 138
Description Sequence SEQ ID No. TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC
Description Sequence SEQ ID No. CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCGCAGCAGCAAAGAGAAAGTACTTCGCAGCATAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS10 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG
AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA 139
Description Sequence SEQ ID No. GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA
Description Sequence SEQ ID No. AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCAGAGCAGCAAAGAGAAAGGCATTCGCAGCATAG open reading frame ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT to cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG NLS11 with TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA early and stop codons GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC
CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCCACCA 140
Description Sequence SEQ ID No. GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT
Description Sequence SEQ ID No. ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA
GCAGAGCAGCAAAGAGAAAGTACTTCGCAGTCTAG ORF cas9 CCTAAGAAAAAGCGGAAGGTCGACGGGGATAAGAAGTACTCAATCGGGCTGG using codons typically ATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTG with high expression CCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCATCAAGA AAAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGAC CCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCAT humans (without codons CTGCTATCTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCGACGACAGCT start or stop; TCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATGA ACGGCATCCTATCTTTGGAAACATCGTCGACGAAGTGGCGTACCACGAAAAGT suitable for inclusion in ACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGC sequence encoding the CGACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGAC ACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTT TTCATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAA fusion protein) TGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTC 141
Description Sequence SEQ ID No. GCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGG ACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTCAAGT CCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTA CGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGAT CTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCT GCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAG CGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGAC AGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAATGGG TACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCA TCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCT GAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATT CCCCACCAGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAG GACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGAC GTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTC GCGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAGGA AGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAACGAATGACCAACT TCGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTAC GAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGG AATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGAT CTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACT ACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGTGGAGGA CAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGG ACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGT CCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAG ACCTACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCG GTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGAT AAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAA TCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACA TCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGC GAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCAAACTGTGAAG GTGGTCGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATC GTGATTGAAATGGCCCGAGAAAACCAGACTACCCCAGAAGGGCCAGAAAAACT CCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCC AGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCT CTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGAGCTG GACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTT TCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGAAC
Description Sequence SEQ ID No. AGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGA ATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGAC AATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGAT TCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCA GATCTTGGACTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATCC GGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAA GGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATG ACGCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCTAAA CTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGA TGATAGCCAAGTCCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTT TACTCAAACATCATGAACTTTTTCAAGACTGAAATTACCTGGCCAATGGAGAA ATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGTGT GGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCA AGTCAATATTGTGAAAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAAT CGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTG GGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTC CTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCA AAGAGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCC GATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATC ATCAAACTCCCCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCAT GCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGC AAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGTC ACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATTAT CTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGC CGACGCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGATAAG CCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCT GGGAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCGCAAAAGAT ACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAAGCATCAC
TGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGAT ORF cas9 GACAAGAAGTACTCTATCGGTTTGGACATCGGTACCAACTCTGTCGGTTGGGC using codons half-life of CGTCATCACCGACGAATACAAGGTCCCATCTAAGAAGTTCAAGGTCTTGGGTA long ACACCGACAGACACTCTATCAAGAAGAACTTGATCGGTGCCTTGTTGTTCGAC TCTGGTGAAACCGCCGAAGCCACCAGATTGAAGAGAACCGCCAGAAGAAGAT Table 4 (no ACACCAGAAGAAAGAACAGAATCTGCTACTTGCAAGAAATCTTCTCTAACGAA start codon or stop ATGGCCAAGGTCGACGACTCTTTCTTCCACAGATTGGAAGAATCTTTCTTGGT; CGAAGAAGACAAGAAGCACGAAAGACACCCAATCTTCGGTAACATCGTCGAC suitable for GAAGTCGCCTACCACGAAAAGTACCCAACCATCTACCACTTGAGAAAGAAGTT 142
Description Sequence SEQ ID NO GGTCGACTCTACCGACAAGGCCGACTTGAGATTGATCTACTTGGCCTTGGCC inclusion in the coding sequence CACATGATCAAGTTCAGAGGTCACTTCTTGATCGAAGGTGACTTGAACCCAGA CAACTCTGACGTCGACAAGTTGTTCATCCAATTGGTCCAAACCTACAACCAATT GTTCGAAGAAAACCCAATCAACGCCTCTGGTGTCGACGCCAAGGCCATCTTG fusion protein) TCTGCCAGATTGTCTAAGAGCAGAAGATTGGAAAACTTGATCGCCCAATTGCC AGGTGAAAAGAAGAACGGTTTGTTCGGTAACTTGATCGCCTTGTCTTTGGGTT TGACCCCAAACTTCAAGTCTAACTTCGACTTGGCCGAAGACGCCAAGTTGCAA TTGTCTAAGGACACCTACGACGACGACTTGGACAACTTGTTGGCCCAAATCGG TGACCAATACGCCGACTTGTTCTTGGCCGCCAAGAACTTGTCTGACGCCATCT TGTTGTCTGACATCTTGAGAGTCAACACCGAAATCACCAAGGCCCCATTGTCT GCCTCTATGATCAAGAGATACGACGAACACCCACCAAGACTTGACCTTGTTGAA GGCCTTGGTCAGACAACAATTGCCAGAAAAGTACAAGGAAATCTTCTTCGACC AATCTAAGAACGGTTACGCCGGTTACATCGACGGTGGTGCCTCTCAAGAAGA ATTCTACAAGTTCATCAAGCCAATCTTGGAAAAGATGGACGGTACCGAAGAAT TGTTGGTCAAGTTGAACAGAGAAGACTTGTTGAGAAAGCAAAGAACCTTCGAC AACGGTTCTATCCCACACCAAATCCACTTGGGTGAATTGCACGCCATCTTGAG AAGACAAGAAGACTTCTACCCATTCTTGAAGGACAACAGAGAAAAGATCGAAA AGATCTTGACCTTCAGAATCCCATACTACGTCGGTCCATTGGCCAGAGGTAAC AGCAGATTCGCCTGGATGACCAGAAAGTCTGAAGAAACCATCACCCCATGGA ACTTCGAAGAAGTCGTCGACAAGGGTGCCTCTGCCCAATCTTTCATCGAAAGA ATGACCAACTTCGACAAGAACTTGCCAAACGAAAAGGTCTTGCCAAAGCACTC TTTGTTGTACGAATACTTCACCGTCTACAACGAATTGACCAAGGTCAAGTACGT CACCGAAGGTATGAGAAAGCCAGCCTTCTTGTCTGGTGAAAAAAGAAGGCC ATCGTCGACTTGTTGTTCAAGACCAACAGAAAGGTCACCGTCAAGCAATTGAA GGAAGACTACTTCAAGAAGATCGAATGCTTCGACTCTGTCGAAATCTCTGGTG TCGAAGACAGATTCAACGCCTCTTTGGGTACCTACCACGACTTGTTGAAGATC ATCAAGGACAAGGACTTCTTGGACAACGAAGAAAACGAAGACATCTTGGAAGA CATCGTCTTGACCTTGACCTTGTTCGAAGACAGAGAAATGATCGAAGAAAGAT TGAAGACCTACGCCCACTTGTTCGACGACAAGGTCATGAAGCAATTGAAGAGA AGAAGATACACCGGTTGGGGTAGATTGAGCAGAAAGTTGATCAACGGTATCA GAGACAAGCAATCTGGTAAGACCATCTTGGACTTCTTGAAGTCTGACGGTTTC GCCAACAGAAACTTCATGCAATTGATCCACGACGACTCTTTGACCTTCAAGGA AGACATCCAAAGGCCCAAGTCTCTGGTCAAGGTGACTCTTTGCACGAACACA TCGCCAACTTGGCCGGTTCTCCAGCCATCAAGAAGGGTATCTTGCAAACCGT CAAGGTCGTCGACGAATTGGTCAAGGTCATGGGTAGACACAAGCCAGAAAAC ATCGTCATCGAAATGGCCAGAGAAAACCAAACCACCCAAAAGGGTCAAAAAGAA CAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGTATCAAGGAATTGGGTTCT
Description Sequence SEQ ID No. CAAATCTTGAAGGAACACCCAGTCGAAAAACCCCAATTGCAAAACGAAAAGTT GTACTTGTACTACTTGCAAAACGGTAGAGACATGTACGTCGACCAAGAATTGG ACATCAACAGATTGTCTGACTACGACGTCGACCACATCGTCCCACAATCTTTC TTGAAGGACGACTCTATCGACAACAAGGTCTTGACCAGATCTGACAAGAACAG AGGTAAGTCTGACAACGTCCCATCTGAAGAAGTCGTCAAGAAGATGAAGAACT ACTGGAGACAATTGTTGAACGCCAAGTTGATCACCCAAAGAAAGTTCGACAAC TTGACCAAGGCCGAAAGAGGTGGTTTGTCTGAATTGGACAAGGCCGGTTTCA TCAAGAGACAATTGGTCGAAAACCAGACAAATCACCAAGCACGTCGCCCAAATC TTGGACAGCAGAATGAACACCAAGTACGACGAAAACGACAAGTTGATCAGAG AAGTCAAGGTCATCACCTTGAAGTCTAAGTTGGTCTCTGACTTCAGAAAGGAC TTCCAATTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCCCACGACGC CTACTTTGAACGCCGTCGTCGGTACCGCCTTGATCAAGAAGTACCCAAAGTTG GAATCTGAATTCGTCTACGGTGACTACAAGGTCTACGACGTCAGAAAGATGAT CGCCAAGTCTGAACAAGAAATCGGTAAGGCCACCGCCAAGTACTTCTTCTACT CTAACATCATGAACTTCTTCAAGACCGAAATCACCTTGGCCAACGGTGAAATC AGAAAAGAGACCATTGATCGAAACCAACGGTGAAACCGGTGAAATCGTCTGGG ACAAGGGTAGAGACTTCGCCACCGTCAGAAAGGTCTTGTCTATGCCACAAGT CAACATCGTCAAGAAGACCGAAGTCCAAAACCGGTGGTTTCTCTAAGGAATCTA TCTTGCCAAAGAGAAACTCTGACAAGTTGATCGCCAGAAAGAAGGACTGGGA CCCAAAGAAGTACGGTGGTTTCGACTCTCCAACCGTCGCCTACTCTGTCTTGG TCGTCGCCAAGGTCGAAAAGGGTAAGTCTAAGAAGTTGAAGTCTGTCAAGGA ATTGTTGGGTATCACCATCATGGAAAGATCTTCTTTCGAAAAGAACCCAATCGA CTTCTTGGAAGCCAAGGGTTACAAGGAAGTCAAGAAGGACTTGATCATCAAGT TGCCAAAGTACTCTTTGTTCGAATTGGAAAACGGTAGAAAGAGAATGTTGGCC TCTGCCGGTGAATTGCAAAAGGGTAACGAATTGGCCTTGCCATCTAAGTACGT CAACTTCTTGTACTTGGCCTCTCACTACGAAAAGTTGAAGGGTTCTCCAGAAG ACAACGAAACAAAAGCAATTGTTCGTCGAACACACACAAGCACTACTTGGACGAA ATCATCGAACAAATCTCTGAATTCTCTAAGAGAGTCATCTTGGCCGACGCCAA CTTGGACAAGGTCTTGTCTGCCTACAACAAGCACAGAGACAAGCCAATCAGA GAAAAAGCCGAAAACATCATCCACTTGTTCACCTTGACCAACTTGGGTGCCCC AGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATACACCTCTA CCAAGGAAGTCTTGGACGCCACTTGATCCACCAATCTATCACCGGTTTGTAC GAAACCAGAATCGACTTGTCTCAATTGGGTGGTGACGGTGGTGGTTCTCCAAA GAAGAAGAGAAAGGTC
Description Sequence SEQ ID No. ORF cas9 GATAAAAAATATTCTATTGGTTTAGATATTGGTACTAATTCTGTTGGTTGGGCT using codons GTTATTACTGATGAATATAAAGTTCCTTCTAAAAAATTTAAAGTTTTAGGTAATA U-rich CTGATCGTCATTCTATTAAAAAAAATTTAATTGGTGCTTTATTATTTGATTCTGG Table 4 (no TGAAACTGCTGAAGCTACTCGTTTAAAACGTACTGCTCGTCGTCGTTATACTC start codon or stop GTCGTAAAAATCGTATTTGTTATTTACAAGAAATTTTTTCTAATGAAATGGCTAA; AGTTGATGATTCTTTTTTTCATCGTTTAGAAGAATCTTTTTTAGTTGAAGAAGAT AAAAAACATGAACGTCATCCTATTTTTGGTAATATTGTTGATGAAGTTGCTTATC suitable for inclusion in ATGAAAAATATCCTACTATTTATCATTTACGTAAAAAATTAGTTGATTCTACTGA sequence encoding the TAAAGCTGATTTACGTTTAATTTATTTAGCTTTAGCTCATATGATTAAATTTCGT GGTCATTTTTTAATTGAAGGTGATTTAAATCCTGATAATTCTGATGTTGATAAAT TATTTATTCAATTAGTTCAAACTTATAATCAATTATTTGAAGAAAATCCTATTAAT fusion protein) GCTTCTGGTGTTGATGCTAAAGCTATTTTATCTGCTCGTTTATCTAAATCTCGT CGTTTAGAAAATTTAATTGCTCAATTACCTGGTGAAAAAAAAAATGGTTTATTTG GTAATTTAATTGCTTTATCTTTAGGTTTAACTCCTAATTTTAAATCTAATTTTGAT TTAGCTGAAGATGCTAAATTACAATTATCTAAAGATACTTATGATGATGATTTAG ATAATTTATTAGCTCAAATTGGTGATCAATATGCTGATTTATTTTTAGCTGCTAA AAATTTATCTGATGCTATTTTATTATCTGATATTTTACGTGTTAATACTGAAATTA CTAAAGCTCCTTTATCTGCTTCTATGATTAAACGTTATGATGAACATCATCAAG ATTTAACTTTATTAAAAGCTTTAGTTCGTCAACAATTACCTGAAAAATATAAAGA AATTTTTTTTGATCAATCTAAAAATGGTTATGCTGGTTATATTGATGGTGGTGCT TCTCAAGAAGAATTTTATAAATTTATTAAACCTATTTTAGAAAAAATGGATGGTA CTGAAGAATTATTAGTTAAATTAAATCGTGAAGATTTATTACGTAAACAACGTAC TTTTGATAATGGTTCTATTCCTCATCAAATTCATTTAGGTGAATTACATGCTATT TTACGTCGTCAAGAAGATTTTTATCCTTTTTTAAAAGATAATCGTGAAAAATTG AAAAAATTTTAACTTTTCGTATTCCTTATTATGTTGGTCCTTTAGCTCGTGGTAA TTCTCGTTTTGCTTGGATGACTCGTAAATCTGAAGAAACTATTACTCCTTGGAA TTTTGAAGAAGTTGTTGATAAAGGTGCTTCTGCTCAATCTTTTATTGAACGTAT GACTAATTTTGATAAAAATTTACCTAATGAAAAAGTTTTACTAAACATTCTTTA TTATATGAATATTTTACTGTTTATAATGAATTAACTAAAGTTAAATATGTTACTGA AGGTATGCGTAAACCTGCTTTTTTATCTGGTGAACAAAAAAAAGCTATTGTTGA TTTATTATTTAAAACTAATCGTAAAGTTACTGTTAAACAATTAAAAGAAGATTATT TTAAAAAAATTGAATGTTTTGATTCTGTTGAAATTTCTGGTGTTGAAGATCGTTT TAATGCTTCTTTAGGTACTTATCATGATTTATTAAAAATTATTAAAGATAAAGATT TTTTAGATAATGAAGAAAATGAAGATATTTTAGAAGATATTGTTTTAACTTTAAC TTTATTTGAAGATCGTGAAATGATTGAAGAACGTTTAAAAACTTATGCTCATTTA
TTTGATGATAAAGTTATGAAACAATTAAAACGTCGTCGTTATACTGGTTGGGGT CGTTTATCTCGTAAATTAATTAATGGTATTCGTGATAAACAATCTGGTAAAACTA 143
Description Sequence SEQ ID No. TTTTAGATTTTTTAAAATCTGATGGTTTTGCTAATCGTAATTTTATGCAATTAATT CATGATGATTCTTTAACTTTTAAAGAAGATATTCAAAAGCTCAAGTTTCTGGTC AAGGTGATTCTTTACATGAACATATTGCTAATTTAGCTGGTTCTCCTGCTATTA AAAAAAGGTATTTTACAAACTGTTAAAGTTGTTGATGAATTAGTTAAAGTTATGG GTCGTCATAAACCTGAAAATATTGTTATTTGAAATGGCTCGTGAAAATCAAACTA CTCAAAAAGGTCAAAAAAATTCTCGTGAACGTATGAACGTATTGAAGAAGGT ATTAAAGAATTAGGTTCTCAAATTTTAAAAGAACATCCTGTTGAAAATACTCAAT TACAAAATGAAAAATTATATTTATATTATTTACAAAATGGTCGTGATATGTATGT TGATCAAGAATTAGATATTAATCGTTTATCTGATTATGATGTTGATCATATTGTT CCTCAATCTTTTTTAAAAGATGATTCTATTGATAATAAAGTTTTAACTCGTTCTG ATAAAAAATCGTGGTAAATCTGATAATGTTCCTTCTGAAGAAGTTGTTAAAAAAAA TGAAAAATTATTGGCGTCAATTATTAAATGCTAAATTAATTACTCAACGTAAATT TGATAATTTAACTAAAGCTGAACGTGGTGGTTTATCTGAATTAGATAAAGCTGG TTTTATTAAACGTCAATTAGTTGAAACTCGTCAAATTACTAAACATGTTGCTCAA ATTTTAGATTCTCGTATGAATACTAAATATGATGAAAATGATAAATTAATTCGTG AAGTTAAAGTTATTACTTTAAAATCTAAATTAGTTTCTGATTTTCGTAAAGATTTT CAATTTTATAAAGTTCGTGAAATTAATAATTATCATCATGCTCATGATGCTTATT TAAATGCTGTTGTTGGTACTGCTTTAATTAAAAAATATCCTAAATTAGAATCTGA ATTTGTTTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAATCT GAACAAGAAATTGGTAAAGCTACTGCTAAATATTTTTTTTATTCTAATATTATGA ATTTTTTTAAAACTGAAATTACTTTAGCTAATGGTGAAATTCGTAAACGTCCTTT AATTGAAACTAATGGTGAAACTGGTGAAATTGTTTGGGATAAAGGTCGTGATTT TGCTACTGTTCGTAAAGTTTTATCTATGCCTCAAGTTAATATTGTTAAAAAAACT GAAGTTCAAACTGGTGGTTTTTCTAAAGAATCTATTTTACCTAAACGTAATTCT GATAAATTAATTGCTCGTAAAAAAGATTGGGATCCTAAAAAATATGGTGGTTTT GATTCTCCTACTGTTGCTTATTCTGTTTTAGTTGTTGCTAAAGTTGAAAAAAGGT AAATCTAAAAAATTAAAATCTGTTAAAGAATTATTAGGTATTACTATTATGGAAC GTTCTTCTTTTGAAAAAAATCCTATTGATTTTTTAGAAGCTAAAGGTTATAAAGA AGTTAAAAAAGATTTAATTATTAAATTACCTAAATATTCTTTATTTGAATTAGAAA ATGGTCGTAAACGTATGTTAGCTTCTGCTGGTGAATTACAAAAAGGTAATGAAT TAGCTTTACCTTCTAAATATGTTAATTTTTTATATTTAGCTTCTCATTATGAAAA TTAAAAGGTTCTCCCTGAAGATAATGAACAAAAACAATTATTTGTTGAACAACAT AAACATTATTTAGATGAAATTATTGAACAAATTTCTGAATTTTCTAAACGTGTTA TTTTAGCTGATGCTAATTTAGATAAAGTTTTATCTGCTTATAATAAACATCGTGA TAAACCTATTCGTGAACAAGCTGAAAATATTATTCATTTATTTACTTTAACTAAT TTAGGTGCTCCTGCTGCTTTTAAATATTTTGATACTACTATTGATCGTAAACGTT ATACTTCTACTAAAGAAGTTTTAGATGCTACTTTAATTCATCAATCTATTACTGG
Description Sequence SEQ ID No. TTTATATGAAACTCGTATTGATTTATCTCAATTAGGTGGTGATGGTGGTGGTTC
TCCTAAAAAAAAACGTAAAGTT ORF cas9 GACAAAAAATACTCCATCGGCCTCGACATCGGCACCAACTCCGTCGGCTGGG using codons CCGTCATCACCGACGAATACAAAGTCCCCTCCAAAAAATTCAAAGTCCTCGGC G below the AACACCGACAGACACTCCATCAAAAAAAACCTCATCGGCGCCCTCCTCTTCGA Table 4 (no CTCCGGCGAAACCGCCGAAGCCACCAGACTCAAAAGAACCGCCAGAAGAAGA start codon or stop TACACCAGAAGAAAAAACAGAATCTGCTACCTCCAAGAAATCTTCTCCAACGA; AATGGCCAAAGTCGACGACTCCTTCTTCCACAGACTCGAAGAATCCTTCCTCG TCGAAGAAGACAAAAAACACGAAAGACACCCCATCTTCGGCAACATCGTCGAC suitable for inclusion in GAAGTCGCCTACCACGAAAAATACCCCACCATCTACCACCTCAGAAAAAAACT sequence encoding the CGTCGACTCCACCGACAAAGCCGACCTCAGACTCATCTACCTCGCCCTCGCC CACATGATCAAATTCAGAGGCCACTTCCTCATCGAAGGCGACCTCAACCCCGA CAACTCCGACGTCGACAAACTCTTCATCCAACTCGTCCAAACCTACAACCAAC fusion protein) TCTTCGAAGAAAACCCCATCAACGCCTCCGGCGTCGACGCCAAAGCCATCCT CTCCGCCAGACTCTCCAAATCCAGAAGACTCGAAAACCTCATCGCCCAACTCC CCGGCGAAAAAAAAAAACGGCCTTCTGGCAACCTCATCGCCCTCTCCCTCGG CCTCACCCCCAACTTCAAATCCAACTTCGACCTCGCCGAAGACGCCAAACTCC AACTCTCCAAAGACACCTACGACGACGACCTCGACAACCTCCTCGCCCAAATC GGCGACCAATACGCCGACCTCTTCCTCGCCGCCAAAAACCTCTCCGACGCCA TCCTCCTCTCCGACATCCTCAGAGTCAACACCGAAATCACCAAAGCCCCCCTC TCCGCCTCCATGATCAAAAAGATACGACGAACACCACCAAGACCTCACCCTCCT CAAAGCCCTCGTCAGACAACAACTCCCCGAAAAATACAAAGAAAATCTTCTTCG ACCAATCCAAAAACGGCTACGCCGGCTACATCGACGGCGGCGCCTCCCAAGA AGAATTCTACAAATTCATCAAACCCATCCTCGAAAAAATGGACGGCACCGAAG AACTCCCTGTCAAACTCAACAGAGAAGACCTCCTCAGAAAACAAAGAACCTTC GACAACGGCTCCATCCCCCACCAAATCCACCTCGGCGAACTCCACGCCATCC TCAGAAGACAAGAAGACTTCTACCCCTTCCTCAAAGACAACAGAGAAAAAATTC GAAAAAATCCTCACCTTCAGAATCCCCTACTACGTCGGCCCCCTCGCCAGAG GCAACTCCAGATTCGCCTGGATGACCAGAAAATCCGAAGAAACCATCACCCC CTGGAACTTCGAAGAAGTCGTCGACAAAGGCGCCTCCGCCCAATCCTTCATC GAAAGAATGACCAACTTCGACAAAAACCTCCCCAACGAAAAAAGTCCTCCCCAA ACACTCCCTCCTCTACGAATACTTCACCGTCTACAACGAACTCACCAAAGTCA
AATACGTCACCCGAAGGCATGAGAAAACCCGCCTTCCTCTCCGGCGAACAAAA AAAAGCCATCGTCGACCTCCTCTTCAAAACCAACAGAAAAGTCACCGTCAAAC 144
Description Sequence SEQ ID No. AACTCAAAGAAGACTACTTCAAAAAAAATCGAATGCTTCGACTCCGTCGAAATCT CCGGCGTCGAAGACAGATTCAACGCCTCCCTCGGCACCTACCACGACCTCCT CAAAATCATCAAAGACAAAGACTTCCTGCAACGAAGAAAACGAAGACATCC TCGAAGACATCGTCCTCACCCTCACCCTCTTCGAAGACAGAGAAATGATCGAA GAAAGACTCAAAACCTACGCCCACCTCTTCGACGACAAAGTCATGAAACAACT CAAAAGAAGAAGATACACCGGCTGGGGCAGACTCTCCAGAAAACTCATCAAC GGCATCAGAGACAAACAATCCGGCAAAACCATCCCTGACTTCCTCAAATCCGA CGGCTTCGCCAAACAGAAACTTCATGCAACTCATCCACGACGACTCCCTCACCT TCAAAGAAGACATCCAAAAAGCCCAAGTCTCCGGCCAAGGCGACTCCCTCCA CGAACACATCGCCAACCTCGCCGGCTCCCCCGCCATCAAAAAAGGCATCCTC CAAACCGTCAAAGTCGTCGACGAACTCGTCAAAGTCATGGGCAGACACAAAC CCGAAAAACATCGTCATCGAAATGGCCAGAGAAAACCAAACCACCCAAAAAGG CCAAAAAAACTCCAGAGAAAGAATGAAAAAGAATCGAAGAAGGCATCAAAGAAC TCGGCTCCCAAATCCTCAAAGAACACCCCGTCGAAAACACCCAACTCCAAAAC GAAAAACTCTACCTCTACTACCTCCAAAACGGCAGAGACATGTACGTCGACCA AGAACTCGACATCAACAGACTCTCCGACTACGACGTCGACCACATCGTCCCC CAATCCTTCCTCAAAGACGACTCCATCGACAACAAAGTCCTCACCAGATCCGA CAAAAAACAGAGGCAAATCCGACAACGTCCCCTCCGAAGAAGTCGTCAAAAAAAA TGAAAAACTACTGGAGACAACTCCTCAACGCCAAACTCATCACCCAAAGAAAA TTCGACAACCTCACCAAAGCCGAAAGAGGCGGCCTCTCCGAACTCGACAAAG CCGGCTTCATCAAAAGACAACTCGTCGAAACCAGACAAATCACCAAACACGTC GCCCAAATCCTCGACTCCAGAATGAAACCCAAATACGACGAAAACGACAAACT CATCAGAGAAGTCAAAGTCATCACCCTCAAAATCCAAACTCGTCTCCGACTTCA GAAAAGACTTCCAATTCTACAAAGTCAGAGAAATCAACAACTACCACCACGCC CACGACGCCTACCTCAACGCCGTCGTCGGCACCGCCCTCATCAAAAAATACC CCAAACTCGAATCCGAATTCGTCTACGGCGACTACAAAGTCTACGACGTCAGA AAAATGATCGCCAAATCCGAACAAGAAATCGGCAAAGCCACCGCCAAATACTT CTTCTACTCCAACATCATGAACTTCTTCAAAACCGAAATCACCCTCGCCAACG GCGAAATCAGAAAAAGACCCCTCATCGAAACCAACGGCGAAACCGGCGAAAT CGTCTGGGACAAAGGCAGAGACTTCGCCACCGTCAGAAAAGTCCTCTCCATG CCCCAAGTCAACATCGTCAAAAAACCGAAGTCCAAAACCGGCGGCTTCTCCAA AGAATCCATCCTCCCCAAAAGAAACTCCGACAAACTCATCGCCAGAAAAAAAG ACTGGGACCCCAAAAAAATACGGCGGCTTCGACTCCCCCACCGTCGCCTACTC CGTCCTCGTCGTCGCCAAAGTCGAAAAAGGCAAATCCAAAAAACTCAAATCCG TCAAAGAACTCCCTCGGCATCACCATCATGGAAAGATCCTCCTTCGAAAAAAAC CCCATCGACTTCCTCGAAGCCAAAGGCTACAAAGAAGTCAAAAAAAGACCTCAT CATCAAACTCCCCAAATACTCCCTCTTCGAACTCGAAAACGGCAGAAAAAGAA
Description Sequence SEQ ID No. TGCTCGCCTCCGCCGGCGAACTCCAAAAAGGCAACGAACTCGCCCTCCCCTC CAAATACGTCAACTTCCTCTACCTCGCCTCCCACTACGAAAAACTCAAAGGCT CCCCCGAAGACAACGAACAAAAAACAACTCTTCGTCGAACAACACAAACACTAC CTCGACGAAATCATCGAACAAATCTCCGAATTCTCCAAAAGAGTCATCCTCGC CGACGCCAACCTCGACAAAGTCCTCTCCGCCTACAACAAACACAGAGACAAA CCCATCAGAGAACAAGCCGAAAACATCATCCACCTCTTCACCCTCACCAACCT CGGCGCCCCCGCCGCCTTCAAATACTTCGACACCACCATCGACAGAAAAAGA TACACCTCCACCAAAGAAGTCCTCGACGCCACCCTCATCCACCAATCCATCAC CGGCCTCTACGAAAACCAGAATCGACCTCTCCCAACTCGGCGGCGACGGCGG
CGGCTCCCCCAAAAAAAAAAGAAAAGTC ORF cas9 GATAAGAAGTATAGTATTGGATTGGATATTGGAACAAATAGTGTGGGATGGGC using codons TGTGATTACAGATGAGTATAAGGTGCCTAGTAAGAAGTTTAAGGTGTTGGGAA C below the ATACAGATAGACATAGTATTAAGAAGAATTTGATTGGAGCTTTGTTGTTTGATA Table 4 (no GTGGAGAGACAGCTGAGGCTACAAGATTGAAGAGAACAGCTAGAAGAAGATA start codon or stop TACAAGAAGAAAGAATAGAATTTGTTATTTGCAGGAGATTTTTAGTAATGAGAT; GGCTAAGGTGGATGATAGTTTTTTTCATAGATTGGAGGAGAGTTTTTTGGTGG AGGAGGATAAGAAGCATGAGAGACATCCTATTTTTGGAAATATTGTGGATGAG suitable for inclusion in GTGGCTTATCATGAGAAGTATCCTACAATTTATCATTTGAGAAAGAAGTTGGTG sequence encoding the GATAGTACAGATAAGGCTGATTTGAGATTGATTTATTTGGCTTTGGCTCATATG ATTAAGTTTAGAGGACATTTTTTGATTGAGGGAGATTTGAATCCTGATAATAGT GATGTGGATAAGTTGTTTATTCAGTTGGTGCAGACATATAATCAGTTGTTTGAG fusion protein) GAGAATCCTATTAATGCTAGTGGAGTGGATGCTAAGGCTATTTTGAGTGCTAG ATTGAGTAAGAGTAGAAGATTGGAGAATTTGATTGCTCAGTTGCCTGGAGAGA AGAAGAATGGATTGTTTGGAAATTTGATTGCTTTGAGTTTGGGATTGACACCTA ATTTTAAGAGTAATTTTGATTTGGCTGAGGATGCTAAGTTGCAGTTGAGTAAGG ATACATATGATGATGATTTGGATAATTTGTTGGCTCAGATTGGAGATCAGTATG CTGATTTGTTTTTGGCTGCTAAGAATTTGAGTGATGCTATTTTGTTGAGTGATA TTTTGAGAGTGAATACAGAGATTACAAAGGCTCCTTTGAGTGCTAGTATGATTA AGAGATATGATGAGCATCATCAGGATTTGACATTGTTGAAGGCTTTGGTGAGA CAGCAGTTGCCTGAGAAGTATAAGGAGATTTTTTTTGATCAGAGTAAGAATGG ATATGCTGGATATATTGATGGAGGAGCTAGTCAGGAGGAGTTTTATAAGTTTAT TAAGCCTATTTTGGAGAAGATGGATGGAACAGAGGAGTTGTTGGTGAAGTTGA ATAGAGAGGATTTGTTGAGAAAGCAGAGAACATTTGATAATGGAAGTATTCCT CATCAGATTCATTTGGGAGAGTTGCATGCTATTTTGAGAAGACAGGAGGATTT TTATCCTTTTTTGAAGGATAATAGAGAGAAGATTGAGAAGATTTTGACATTTAG
AATTCCTTATTATGTGGGACCTTTGGCTAGAGGAAATAGTAGATTTGCTTGGAT GACAAGAAAGAGTGAGGAGACAATTACACCTTGGAATTTTGAGGAGGTGGTG 145
Description Sequence SEQ ID No. GATAAGGGAGCTAGTGCTCAGAGTTTTATTGAGAGAATGACAAATTTTGATAA GAATTTGCCTAATGAGAAGGTGTTGCCTAAGCATAGTTTGTTGTATGAGTATTT TACAGTGTATAATGAGTTGACAAAGGTGAAGTATGTGACAGAGGGAATGAGAA AGCCTGCTTTTTTGAGTGGAGAGCAGAAGAAGGCTATTGTGGATTTGTTGTTT AAGACAAATAGAAAGGTGACAGTGAAGCAGTTGAAGGAGGATTATTTTAAGAA GATTGAGTGTTTTGATAGTGTGGAGATTAGTGGAGTGGAGGATAGATTTAATG CTAGTTTGGGAACATATCATGATTTGTTGAAGATTATTAAGGATAAGGATTTTTT GGATAATGAGGAGAATGAGGATATTTTGGAGGATATTGTGTTGACATTGACAT TGTTTGAGGATAGAGAGATGATTGAGGAGAGATTGAAGACATATGCTCATTTG TTTGATGATAAGGTGATGAAGCAGTTGAAGAGAAGAAGATATACAGGATGGGG AAGATTGAGTAGAAAGTTGATTAATGGAATTAGAGATAAGCAGAGTGGAAAGA CAATTTTGGATTTTTTGAAGAGTGATGATTTGCTAATAGAAATTTTATGCAGTT GATTCATGATGATAGTTTGACATTTAAGGAGGATATTCAGAAGGCTCAGGTGA GTGGACAGGGAGATAGTTTGCATGAGCATATTGCTAATTTGGCTGGAAGTCCT GCTATTAAGAAGGGAATTTTGCAGACAGTGAAGGTGGTGGATGAGTTGGTGA AGGTGATGGGAAGACATAAGCCTGAGAATATTGTGATTGAGATGGCTAGAGA GAATCAGACAACACAGAAGGGACAGAAGAATAGTAGAGAGAGAATGAAGAGA ATTGAGGAGGGAATTAAGGAGTTGGGAAGTCAGATTTTGAAGGAGCATCCTGT GGAGAATACACAGTTGCAGAATGAGAAGTTGTATTTGTATTATTTGCAGAATG GAAGAGATATGTATGTGGATCAGGAGTTGGATATTAATAGATTGAGTGATTAT GATGTGGATCATATTGTGCCTCAGAGTTTTTTGAAGGATGATAGTATTGATAAT AAGGTGTTGACAAGAAGTGATAAGAATAGAGGAAAGAGTGATAATGTGCCTAG TGAGGAGGTGGTGAAGAAGATGAAGAATTATTGGAGACAGTTGTTGAATGCTA AGTTGATTACACAGAGAAAGTTTGATAATTTGACAAAGGCTGAGAGAGGAGGA TTGAGTGAGTTGGATAAGGCTGGATTTATTAAGAGACAGTTGGTGGAGACAAG ACAGATTACAAAGCATGTGGCTCAGATTTTGGATAGTAGAATGAATACAAAGTA TGATGAGAATGATAAGTTGATTAGAGAGGTGAAGGTGATTACATTGAAGAGTA AGTTGGTGAGTGATTTTAGAAAGGATTTTCAGTTTTATAAGGTGAGAGAGATTA ATAATTATCATCATGCTCATGATGCTTATTTGAATGCTGTGGTGGGAACAGCTT TGATTAAGAAGTATCCTAAGTTGGAGAGTGAGTTTGTGTATGGAGATTATAAG GTGTATGATGTGAGAAAGATGATTGCTAAGAGTGAGCAGGAGATTGGAAAGG CTACAGCTAAGTATTTTTTTTATAGTAATATTATGAATTTTTTTAAGACAGAGATT ACATTGGCTAATGGAGAGATTAGAAAGAGACCTTTGATTGAGACAAATGGAGA GACAGGAGAGATTGTGTGGGATAAGGGAAGAGATTTTGCTACAGTGAGAAAG GTGTTGAGTATGCCTCAGGTGAATATTGTGAAGAAGACAGAGGTGCAGACAG GAGGATTTAGTAAGGAGAGTATTTTGCCTAAGAGAAATAGTGATAAGTTGATT GCTAGAAAGAAGGATTGGGATCCTAAGAAGTATGGAGGATTTGATAGTCCTAC
Description Sequence SEQ ID No. AGTGGCTTATAGTGTGTTGGTGGTGGCTAAGGTGGAGAAGGGAAAGAGTAAG AAGTTGAAGAGTGTGAAGGAGTTGTTGGGAATTACAATTATGGAGAGAAGTAG TTTTGAGAAGAATCCTATTGATTTTTTGGAGGCTAAGGGATATAAGGAGGTGA AGAAGGATTTGATTATTAAGTTGCCTAAGTATAGTTTGTTTGAGTTGGAGAATG GAAGAAAGAGAATGTTGGCTAGTGCTGGAGAGTTGCAGAAGGGAAATGAGTT GGCTTTGCCTAGTAAGTATGTGAATTTTTTGTATTTGGCTAGTCATTATGAGAA GTTGAAGGGAAGTCCTGAGGATAATGAGCAGAAGCAGTTGTTTGTGGAGCAG CATAAGCATTATTTGGATGAGATTATTGAGCAGATTAGTGAGTTTAGTAAGAGA GTGATTTTGGCTGATGCTAATTTGGATAAGGTGTTGAGTGCTTATAATAAGCAT AGAGATAAGCCTATTAGAGAGCAGGCTGAGAATATTATTCATTTGTTTACATTG ACAAATTTGGGAGCTCCTGCTGCTTTTAAGTATTTTGATACAACAATTGATAGA AAGAGATATACAAGTACAAAGGAGGTGTTGGATGCTACATTGATTCATCAGAG TATTACAGGATTGTATGAGACAAGAATTGATTTGAGTCAGTTGGGAGGAGATG
GAGGAGGAAGTCCTAAGAAGAAGAGAAAGGTG ORF cas9 GACAAGAAGTACTCCATCGGCCTGGACATCGGCACCAACTCCGTGGGCTGGG using codons CCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGG A low CAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTC of Table 4 (no GACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG start codon or stop GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTCC; AACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCCT TCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACAT suitable for inclusion in CGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGG sequence encoding AAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTGG CCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCT GAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACC fusion protein) TACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCCA AGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTGA TCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCG CCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCGA GGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT
CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC 146
Description Sequence SEQ ID No. CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCC AGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGA CAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAA GATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGG AAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGAC AAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAG CACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACG ACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTC CGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTAC CACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATC AAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGT ACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCA
Description Sequence SEQ ID No. CCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCA CCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCG AGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGA AGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGAC CGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTG ATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCC CCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAG TCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGC GGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAA GGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAG CTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAG GGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCT CCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGC TGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTC CGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT CGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCAAGAAGAAGC
GGAAGGTG ORF cas9 GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG A / U low GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT of Table 4 (no CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC start codon or stop GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA; GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA suitable for inclusion in ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT sequence encoding the GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC fusion protein) AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT
GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG 147
Description Sequence SEQ ID No. GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA
Description Sequence SEQ ID No. GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGC
AGCCCCAAGAAGAAGCGGAAGGTG ORF cas9 GACAAGAAGTACTCCATCGGCCTGGACATCGGCACCAACTCCGTGGGCTGGG using codons CCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGGG A low CAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTC of Table 4 with GACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG two sequences C-terminal NLS GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTCC AACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCCT (no codons TCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACAT start or stop; CGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGG suitable for AAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTGG 148
Description Sequence SEQ ID NO inclusion in CCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCT sequence encoding GAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACC TACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCCA AGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTGA fusion protein) TCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCG CCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCGA GGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG
Description Sequence SEQ ID No. CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCC AGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGA CAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAA GATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGG AAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGAC AAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAG CACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACG ACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTC CGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTAC CACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATC AAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGT ACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCA CCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCA CCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCG AGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGA AGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGAC CGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTG ATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCC CCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAG TCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGC GGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAA GGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAG CTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAG GGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCT CCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGC TGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTC CGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT CGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCAAGAAGAAGCG GAAGGTGGACGGCTCCCCCCAAGAAGAAGCGGAAGGTGGACTCCGGC
Description Sequence SEQ ID No. ORF cas9 GACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGG A low GCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT of Table 4 with CGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC two sequences C-terminal NLS GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTC CAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCC (no codons TTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA start or stop; TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCG GAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTG suitable for inclusion in GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC sequence TGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC coding of CTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCC AAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTG fusion) ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATC GCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCG AGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA
GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC 149
Description Sequence SEQ ID No. TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCC AGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGA CAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAA GATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGG AAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGAC AAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAG CACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACG ACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTC CGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTAC CACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATC AAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGT ACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCA CCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCA CCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCG AGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGA AGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGAC CGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTG ATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCC CCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAG TCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGC GGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAA GGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAG CTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAG GGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCT CCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGC TGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTC
Description Sequence SEQ ID No. CGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT CGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCAAGAAGAAGC
GGAAGGTG ORF cas9 GACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCTGG nickase using codons GCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGG The GCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT CGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC below Table 4 (without NLS and without GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTC codons CAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCC start or stop; TTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCG suitable for inclusion in GAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTG sequence encoding GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC CTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCC fusion protein) AAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTG ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATC GCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCG AGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA
CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT 150
Description Sequence SEQ ID No. TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCC AGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGA CAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAA GATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGG AAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGAC AAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAG CACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACG ACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTC CGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTAC CACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATC AAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGT ACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCA CCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCA CCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCG AGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGA AGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGAC CGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTG ATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCC CCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAG
Description Sequence SEQ ID No. TCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGC GGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAA GGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAG CTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAG GGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCT CCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGC TGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTC CGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT
CGACCTGTCCCAGCTGGGCGGCGAC ORF cas9 GACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCTGG nickase using GCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGG codons The GCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT below Table CGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC 4 with two GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTC sequences NLS CAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCC C-terminal TTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA (no codons TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCG start or stop; GAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTG suitable for GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC inclusion in TGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC sequence CTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCC coding AAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTG ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATC protein fusion) GCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCA ACTTCGACCTGGCCG AGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG
GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC 151
Description Sequence SEQ ID No. ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACCACATCGTGCCCC AGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCGA CAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGAA GATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGG AAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGAC AAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAG CACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAACG ACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGTC CGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTAC CACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATC AAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGT ACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCCA CCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATCA
Description Sequence SEQ ID No. CCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCG AGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGA AGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGAC CGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCTG ATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTCC CCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAG TCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGC GGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAA GGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGAG CTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAAG GGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCCT CCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAGC TGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCTC CGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT CGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCAAGAAGAAGCG
GAAGGTGGACGGCTCCCCCAAGAAGAAGCGGAAGGTGGACTCCGGC ORF dCas9 GACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGG A low GCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT of Table 4 (no CGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC start codon or stop GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTC; CAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCC TTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA suitable for inclusion in TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCG sequence encoding the GAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTG GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC fusion protein) CTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCC AAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTG ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATC GCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCG
AGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA 152
Description Sequence SEQ ID No. GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACGCCATCGTGCCC CAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCG ACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGA AGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCG GAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGA CAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAA
Description Sequence SEQ ID No. GCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAAC GACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGT CCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTA CCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGAT CAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTG TACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCC ACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATC ACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGG AAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGA CCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCT GATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTC CCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAA GTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAG CGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACA AGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGA GCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAA GGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCC TCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAG CTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGATCATCGAGCAGATCT CCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT CGACCTGTCCCAGCTGGGCGGCGACGGCGGCGGCTCCCCCAAGAAGAAGC
GGAAGGTG ORF dCas9 GACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGG A low GCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT of Table 4 (no CGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC and without NLS GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTC start or stop codons CAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCC; TTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCG suitable for inclusion in the sequence 153 GAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTG GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC
Description Sequence SEQ ID No. coding of TGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC protein of CTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCC fusion) AAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTG ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATC GCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCG AGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA
Description Sequence SEQ ID No. GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACGCCATCGTGCCC CAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCG ACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGA AGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCG GAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGA CAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAA GCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAAC GACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGT CCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTA CCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGAT CAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTG TACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCC ACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATC ACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGG AAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGA CCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCT GATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTC CCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAA GTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAG CGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACA AGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGA GCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAA GGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCC TCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAG CTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGATCATCGAGCAGATCT CCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT
CGACCTGTCCCAGCTGGGCGGCGAC ORF dCas9 GACAAGAAGTACTCCATCGGCCTGGCCATCGGCACCAACTCCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCTCCAAGAAGTTCAAGGTGCTGG A low GCAACACCGACCGGCACTCCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT of Table 4 with 154 CGACTCCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC
Description Sequence SEQ ID NO GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCTC two sequences C-terminal NLS CAACGAGATGGCCAAGGTGGACGACTCCTTCTTCCACCGGCTGGAGGAGTCC (no codons TTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA start or stop; TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCG GAAGAAGCTGGTGGACTCCACCGACAAGGCCGACCTGCGGCTGATCTACCTG suitable for inclusion in GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC sequence encoding TGAACCCCGACAACTCCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC CTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCTCCGGCGTGGACGCC AAGGCCATCCTGTCCGCCCGGCTGTCCAAGTCCCGGCGGCTGGAGAACCTG fusion protein) ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATC GCCCTGTCCCTGGGCCTGACCCCCAACTTCAAGTCCAACTTCGACCTGGCCG AGGACGCCAAGCTGCAGCTGTCCAAGGACACCTACGACGACGACCTGGACAA CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAA GAACCTGTCCGACGCCATCCTGCTGTCCGACATCCTGCGGGTGAACACCGAG ATCACCAAGGCCCCCCTGTCCGCCTCCATGATCAAGCGGTACGACGAGCACC ACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA AGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACAT CGACGGCGGCGCCTCCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGAC CTGCTGCGGAAGCAGCGGACCTTCGACAACGGCTCCATCCCCCACCAGATCC ACCTGGGGCAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCT TCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCC CTACTACGTGGGCCCCCTGGCCCGGGGCAACTCCCGGTTCGCCTGGATGAC CCGGAAGTCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGA CAAGGGCGCCTCCGCCCAGTCCTTCATCGAGCGGATGACCAACTTCGACAAG AACCTGCCCCAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG GAAGCCCGCCTTCCTGTCCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTC AAGAAGATCGAGTGCTTCGACTCCGTGGAGATCTCCGGCGTGGAGGACCGGT TCAACGCCTCCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAGACC TACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGG TACACCGGCTGGGGCCGGCTGTCCCGGAAGCTGATCAACGGCATCCGGGAC AAGCAGTCCGGCAAGACCATCCTGGACTTCCTGAAGTCCGACGGCTTCGCCA ACCGGAACTTCATGCAGCTGATCCACGACGACTCCCTGACCTTCAAGGAGGA
Description Sequence SEQ ID No. CATCCAGAAGGCCCAGGTGTCCGGCCAGGGCGACTCCCTGCACGAGCACAT CGCCAACCTGGCCGGCTCCCCCGCCATCAAGAAGGGCATCCTGCAGACCGT GAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAA CATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGAA GAACTCCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGG CTCCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGA GAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAG GAGCTGGACATCAACCGGCTGTCCGACTACGACGTGGACGCCATCGTGCCC CAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCCGGTCCG ACAAGAACCGGGGCAAGTCCGACAACGTGCCCTCCGAGGAGGTGGTGAAGA AGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCG GAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGTCCGAGCTGGA CAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAA GCACGTGGCCCAGATCCTGGACTCCCGGATGAACACCAAGTACGACGAGAAC GACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGTCCAAGCTGGTGT CCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTA CCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGAT CAAGAAGTACCCCAAGCTGGAGTCCGAGTTCGTGTACGGCGACTACAAGGTG TACGACGTGCGGAAGATGATCGCCAAGTCCGAGCAGGAGATCGGCAAGGCC ACCGCCAAGTACTTCTTCTACTCCAACATCATGAACTTCTTCAAGACCGAGATC ACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGG AAGGTGCTGTCCATGCCCCAGGTGAACATCGTGAAGAAGACCGAGGTGCAGA CCGGCGGCTTCTCCAAGGAGTCCATCCTGCCCAAGCGGAACTCCGACAAGCT GATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACTC CCCCACCGTGGCCTACTCCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAA GTCCAAGAAGCTGAAGTCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAG CGGTCCTCCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACA AGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACTCCCTGTTCGA GCTGGAGAACGGCCGGAAGCGGATGCTGGCCTCCGCCGGCGAGCTGCAGAA GGGCAACGAGCTGGCCCTGCCCTCCAAGTACGTGAACTTCCTGTACCTGGCC TCCCACTACGAGAAGCTGAAGGGCTCCCCCGAGGACAACGAGCAGAAGCAG CTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGATCATCGAGCAGATCT CCGAGTTCTCCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT GTCCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAG TACTTCGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAGGAGGTGC
Description Sequence SEQ ID No. TGGACGCCACCTGATCCACCAGTCCATCACCGGCCTGTACGAGACCCGGAT CGACCTGTCCCAGCTGGGCGGCGACGGCTCCGGCTCCCCCAAGAAGAAGCG
GAAGGTGGACGGCTCCCCCAAGAAGAAGCGGAAGGTGGACTCCGGC ORF cas9 GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG A / U below the GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT Table 4 with CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC two sequences GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA NLS C-terminal GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA (no codons GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA start or stop; ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT suitable for GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA inclusion in CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC sequence GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC coding AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA fusion) CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG
GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG 155
Description Sequence SEQ ID No. GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT
Description Sequence SEQ ID No. ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCA GCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGG
TGGACAGCGGC ORF cas9 GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG A / U below the GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT Table 4 (no CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC NLS and without GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA start codon GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA or stopped; GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA suitable for ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT inclusion in GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA sequence CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC coding GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC protein AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA fusion) CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG
GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC 156
Description Sequence SEQ ID No. TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA
Description Sequence SEQ ID No. GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC
TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGAC ORF cas9 GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGG nickase GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG codons using A / U GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC below Table 4 (no start codon or stop GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA; GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT suitable for inclusion in GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA sequence encoding the CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA fusion protein) CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC
GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG 157
Description Sequence SEQ ID No. AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG
Description Sequence SEQ ID No. CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGC
AGCCCCAAGAAGAAGCGGAAGGTG ORF cas9 GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGG nickase using GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG codons of A / U GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT below Table CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC 4 with two GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA sequences NLS GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA C-terminal GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA (no codons ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT start or stop; GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA suitable for CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC inclusion in GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA 158 sequence
Description Sequence SEQ ID Coding No. of CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA proteins of CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT fusion) GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA
Description Sequence SEQ ID No. CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCA GCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGG
TGGACAGCGGC ORF of Cas9 GACAAGAAGTACAGCATCGGCCTGGcCATCGGCACCAACAGCGTGGGCTGG nickase using GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG codons of A/U GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT CGACAGCGGCGAGACCCTGCTGGCCGAGACCCTGCTGTT under the CGACAGCGGCGAGACCCTGCTGTT table
Description Sequence SEQ ID NO: 4 (without NLS and without GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA codons GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA start or stop; GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT suitable for inclusion in GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA sequence encoding CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA fusion protein) CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT
Description Sequence SEQ ID No. TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACCA CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA
Description Sequence SEQ ID No. GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC
TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGAC ORF dCas9 GACAAGAAGTACAGCATCGGCCTGGcCATCGGCACCAACAGCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG A / U low GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT of Table 4 (no CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC start codon or stop GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA; GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA suitable for inclusion in ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT sequence encoding the GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC fusion protein) AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG
GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT 160
Description Sequence SEQ ID No. CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC
TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACgcC ATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGA CCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT
Description Sequence SEQ ID No. TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCGGCGGC
AGCCCCAAGAAGAAGCGGAAGGTG ORF dCas9 GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGG using GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG codons of A / U below the GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT Table 4 with CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC two sequences GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA terminal NLS GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA C (without codons beginning of GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA or ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT stop; GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA suitable for CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC inclusion in GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC sequence AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA coding of CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA proteins of CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT) GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAAC TTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCTGCTGAAGGCCCTGGTGCGGCCAGCAGCTGC CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC
GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG 161
Description Sequence SEQ ID No. TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACGC CATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA
Description Sequence SEQ ID No. CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACGGCAGCGGCA GCCCCAAGAAGAAGCGGAAGGTGGACGGCAGCCCCAAGAAGAAGCGGAAGG
TGGACAGCGGC ORF dCas9 GACAAGAAGTACAGCATCGGCCTGGcCATCGGCACCAACAGCGTGGGCTGG using codons GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGG A / U below the GCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTT Table 4 (no CGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC NLS and without GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCA start codon GCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA or stopped; GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCA suitable for ACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCT inclusion in GCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA sequence CCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGC coding of GACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC protein AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGA fusion) CGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAA CCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCT GATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTG GCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTG GACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACA
CCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACG AGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGC 162
Description Sequence SEQ ID No. CCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGG CTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCC ATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGG GAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCC GGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCT GGATGACCCGGAAGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGG TGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACT TCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTA CGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAG GACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGG AGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCG GCTGAAGACTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAG CGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGG CATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAGAGCGAC GGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCC TGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACA AGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGA AGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCA AGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGC
TGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGACgcC ATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGA CCCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAG GTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA TCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGA GCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGC AGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTA CGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAGAG CAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAG
Description Sequence SEQ ID No. ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCA CCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGA CTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATC GGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGA GACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGC CACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGAC CGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAA CAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGG CGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAG GCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGT ACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCG GCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACT TCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACA ACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACACAAGCACTACCTGGACGAGAT CATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAA CCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCG GGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCC CCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCA GCACCAAGGAGGTGCTGGACGCCACCTGATCCACCAGAGCATCACCGGCC
TGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGAC ORF Nme GCCGCCTTCAAGCCCAACTCCATCAACTACATCCTGGGCCTGGACATCGGCA cas9 using codons TCGCCTCCGTGGGCTGGGCCATGGTGGAGATCGACGAGGAGGAGAACCCCA The TCCGGCTGATCGACCTGGGCGTGCGGGTGTTCGAGCGGGCCGAGGTGCCCA AGACCGGCGACTCCCTGGCCATGGCCCGGCGGCTGGCCCGGTCCGTGCGG below Table 4 (no start codon or stop CGGCTGACCCGGCGGCGGGCCCACCGGCTGCTGCGGACCCGGCGGCTGCT GAAGCGGGAGGGCGTGCTGCAGGCCGCCAACTTCGACGAGAACGGCCTGAT; CAAGTCCCTGCCCAACACCCCCTGGCAGCTGCGGGCCGCCGCCCTGGACCG GAAGCTGACCCCCCTGGAGTGGTCCGCCGTGCTGCTGCACCTGATCAAGCAC suitable for inclusion in CGGGGCTACCTGTCCCAGCGGAAGAACGAGGGCGAGACCGCCGACAAGGAG sequence encoding CTGGGCGCCCTGCTGAAGGGCGTGGCCGGCAACGCCCACGCCCTGCAGACC GGCGACTTCCGGACCCCCGCCGAGCTGGCCCTGAACAAGTTCGAGAAGGAG TCCGGCCACATCCGGAACCAGCGGTCCGACTACTCCCACACCTTCTCCCGGA fusion protein) AGGACCTGCAGGCCGAGCTGATCCTGCTGTTCGAGAAGCAGAAGGAGTTCG 163 GCAACCCCCACGTGTCCGGCGGCCTGAAGGAGGGCATCGAGACCCTGCTGA
Description Sequence SEQ ID No. TGACCCAGCGGCCCGCCCTGTCCGGCGACGCCGTGCAGAAGATGCTGGGCC ACTGCACCTTCGAGCCCGCCGAGCCCAAGGCCGCCAAGAACACCTACACCG CCGAGCGGTTCATCTGGCTGACCAAGCTGAACAACCTGCGGATCCTGGAGCA GGGCTCCGAGCGGCCCCTGACCGACACCGAGCGGGCCACCCTGATGGACGA GCCCTACCGGAAGTCCAAGCTGACCTACGCCCAGGCCCGGAAGCTGCTGGG CCTGGAGGACACCGCCTTCTTCAAGGGCCTGCGGTACGGCAAGGACAACGC CGAGGCCCTCCACCCTGATGGAGATGAAGGCCTACCACGCCATCTCCCGGGC CCTGGAGAAGGAGGGCCTGAAGGACAAGAAGTCCCCCCTGAACCTGTCCCC CGAGCTGCAGGACGAGATCGGCACCGCCTTCTCCCTGTTCAAGACCGACGAG GACATCACCGGCCGGCTGAAGGACCGGATCCAGCCCGAGATCCTGGAGGCC CTGCTGAAGCACATCTCCTTCGACAAGTTCGTGCAGATTCCCCTGAAGGCCCT GCGGCGGATCGTGCCCCTGATGGAGCAGGGCAAGCGGTACGACGAGGCCTG CGCCGAGATCTACGGCGACCACTACGGCAAGAAGAACACCGAGGAGAAGAT CTACCTGCCCCCCATCCCCGCCGACGAGATCCGGAACCCCGTGGTGCTGCG GGCCCTGTCCCAGGCCCGGAAGGTGATCAACGGCGTGGTGCGGCGGTACGG CTCCCCCGCCCGGATCCACATCGAGACCGCCCGGGAGGTGGGCAAGTCCTT CAAGGACCGGAAGGAGATCGAGAAGCGGCAGGAGGAGAACCGGAAGGACC GGGAGAAGGCCGCCGCCAAGTTCCGGGAGTACTTCCCCAACTTCGTGGGCG AGCCCAAGTCCAAGGACATCCTGAAGCTGCGGCTGTACGAGCAGCAGCACG GCAAGTGCCTGTACTCCGGCAAGGAGATCAACCTGGGCCGGCTGAACGAGA AGGGCTACGTGGAGATCGACCACCGCCCTGCCCTTCTCCCGGACCTGGGACG ACTCCTTCAACAACAAGGTGCTGGTGCTGGGCTCCGAGAACCAGAACAAGGG CAACCAGACCCCCTACGAGTACTTCAACGGCAAGGACAACTCCCGGGAGTGG CAGGAGTTCAAGGCCCGGGTGGAGACCCTCCGGTTCCCCCGGTCCAAGAAG CAGCGGATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGCGGAAC CTGAACGACACCCGGTACGTGAACCGGTTCCTGTGCCAGTTCGTGGCCGACC GGATGCGGCTGACCGGCAAGGGCAAGAAGCGGGTGTTCGCCTCCAACGGCC AGATCACCAACCTGCTGCGGGGCTTCTGGGGCCTGCGGAAGGTGCGGGCCG AGAAGACCGGCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCCTCCACCG TGGCCATGCAGCAGAAGATCACCCGGTTCGTGCGGTACAAGGAGATGAACGC CTTCGACGGCAAGACCATCGACAAGGAGACCGGCGAGGTGCTGCACCAGAA GACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGATGATCCGG GTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCCGACACCCTG GAGAAGCTGCGGACCCTGCTGGCCGAGAAGCTGTCCTCCCGGCCCGAGGCC GTGCACGAGTACGTGACCCCCTGTTCGTGTCCCGGGCCCCCAACCGGAAG ATGTCCGGCCAGGGCCACATGGAGACCGTGAAGTCCGCCAAGCGGCTGGAC GAGGGCGTGTCCGTGCTGCGGGTGCCCCTGACCCAGCTGAAGCTGAAGGAC
Description Sequence SEQ ID No. CTGGAGAAGATGGTGAACCGGGAGCGGGAGCCCAAGCTGTACGAGGCCCTG AAGGCCCGGCTGGAGGCCCACAAGGACGACCCCGCCAAGGCCTTCGCCGAG CCCTTCTACAAGTACGACAAGGCCGGCAACCGGACCCAGCAGGTGAAGGCC GTGCGGGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGTGCGGAACCACAAC GGCATCGCCGACAACGCCACCATGGTGCGGGTGGACGTGTTCGAGAAGGGC GACAAGTACTACCTGGTGCCCATCTACTCCTGGCAGGTGGCCAAGGGCATCC TGCCCGACCGGGCCGTGGTGCAGGGCAAGGACGAGGAGGACTGGCAGCTG ATCGACGACTCCTTCAACTTCAAGTTCTCCCTGCACCCCAACGACCTGGTGGA GGTGATCACCAAGAAGGCCCGGATGTTCGGCTACTTCGCCTCCTGCCACCGG GGCACCGGCAACATCAACATCCGGATCCACGACCTGGACCACAAGATCGGCA AGAACGGCATCCTGGAGGGCATCGGCGTGAAGACCGCCCTGTCCTTCCAGAA GTACCAGATCGACGAGCTGGGCAAGGAGATCCGGCCCTGCCGGCTGAAGAA GCGGCCCCCCGTGCGGTCCGGCAAGCGGACCGCCGACGGCTCCGAGTTCG
AGTCCCCCAAGAAGAAGCGGAAGGTGGAG ORF Nme GCCGCCTTCAAGCCCAACAGCATCAACTACATCCTGGGCCTGGACATCGGCA cas9 TCGCCAGCGTGGGCTGGGCCATGGTGGAGATCGACGAGGAGGAGAACCCCA codons using A / U TCCGGCTGATCGACCTGGGCGTGCGGGTGTTCGAGCGGGCCGAGGTGCCCA AGACCGGCGACAGCCTGGCCATGGCCCGGCGGCTGGCCCGGAGCGTGCGG below Table 4 (no start codon CGGCTGACCCGGCGGCGGGCCCACCGGCTGCTGCGGACCCGGCGGCTGCT or stop GAAGCGGGAGGGCGTGCTGCAGGCCGCCAACTTCGACGAGAACGGCCTGAT; CAAGAGCCTGCCCAACACCCCCTGGCAGCTGCGGGCCGCCGCCCTGGACCG GAAGCTGACCCCCCTGGAGTGGAGCGCCGTGCTGCTGCACCTGATCAAGCA suitable for inclusion in CCGGGGCTACCTGAGCCAGCGGAAGAACGAGGGCGAGACCGCCGACAAGG sequence encoding the AGCTGGGCGCCCTGCTGAAGGGCGTGGCCGGCAACGCCCACGCCCTGCAGA CCGGCGACTTCCGGACCCCCGCCGAGCTGGCCCTGAACAAGTTCGAGAAGG AGAGCGGCCACATCCGGAACCAGCGGAGCGACTACAGCCACACCTTCAGCC fusion protein) GGAAGGACCTGCAGGCCGAGCTGATCCTGCTGTTCGAGAAGCAGAAGGAGTT CGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGGGCATCGAGACCCTGCT GATGACCCAGCGGCCCGCCCTGAGCGGCGACGCCGTGCAGAAGATGCTGGG CCACTGCACCTTCGAGCCCGCCGAGCCCAAGGCCGCCAAGAACACCTACAC CGCCGAGCGGTTCATCTGGCTGACCAAGCTGAACAACCTGCGGATCCTGGAG CAGGGCAGCGAGCGGCCCCTGACCGACACCGAGCGGGCCACCCTGATGGA CGAGCCCTACCGGAAGAGCAAGCTGACCTACGCCCAGGCCCGGAAGCTGCT GGGCCTGGAGGACACCGCCTTCTTCAAGGGCCTGCGGTACGGCAAGGACAA CGCCGAGGCCAGCACCCTGATGGAGATGAAGGCCTACCACGCCATCAGCCG
GGCCCTGGAGAAGGAGGGCCTGAAGGACAAGAAGAGCCCCCTGAACCTGAG CCCCGAGCTGCAGGACGAGATCGGCACCGCCTTCAGCCTGTTCAAGACCGA 164
Description Sequence SEQ ID No. CGAGGACATCACCGGCCGGCTGAAGGACCGGATCCAGCCCGAGATCCTGGA GGCCCTGCTGAAGCACATCAGCTTCGACAAGTTCGTGCAGATCAGCCTGAAG GCCCTGCGGCGGATCGTGCCCCTGATGGAGCAGGGCAAGCGGTACGACGAG GCCTGCGCCGAGATCTACGGCGACCACTACGGCAAGAAGAACACCGAGGAG AAGATCTACCTGCCCCCCATCCCCGCCGACGAGATCCGGAACCCCGTGGTGC TGCGGGCCCTGAGCCAGGCCCGGAAGGTGATCAACGGCGTGGTGCGGCGG TACGGCCAGCCCCGCCCGGATCCACATCGAGACCGCCCGGGAGGTGGGCAAG AGCTTCAAGGACCGGAAGGAGATCGAGAAGCGGCAGGAGGAGAACCGGAAG GACCGGGAGAAGGCCGCCGCCAAGTTCCGGGAGTACTTCCCCAACTTCGTG GGCGAGCCCAAGAGCAAGGACATCCTGAAGCTGCGGCTGTACGAGCAGCAG CACGGCAAGTGCCTGTACAGCGGCAAGGAGATCAACCTGGGCCGGCTGAAC GAGAAGGGCTACGTGGAGATCGACCACGCCCTGCCCTTCAGCCGGACCTGG GACGACAGCTTCAACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACA AGGGCAACCAGACCCCCTACGAGTACTTCAACGGCAAGGACAACAGCCGGG AGTGGCAGGAGTTCAAGGCCCGGGTGGAGACCAGCCGGTTCCCCCGGAGCA AGAAGCAGCGGATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGC GGAACCTGAACGACACCCGGTACGTGAACCGGTTCCTGTGCCAGTTCGTGGC CGACCGGATGCGGCTGACCGGCAAGGGCAAGAAGCGGGTGTTCGCCAGCAA CGGCCAGATCACCAACCTGCTGCGGGGCTCTGGGGCCTGCGGAAGGTGCG GGCCGAGAACGACCGGCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCA GCACCGTGGCCATGCAGCAGAAGATCACCCGGTTCGTGCGGTACAAGGAGAT GAACGCCTTCGACGGCAAGACCATCGACAAGGAGACCGGCGAGGTGCTGCA CCAGAAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGATG ATCCGGGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCCGAC ACCCTGGAGAAGCTGCGGACCCTGCTGGCCGAGAAGCTGAGCAGCCGGCCC GAGGCCGTGCACGAGTACGTGACCCCCTGTTCGTGAGCCGGGCCCCCAAC CGGAAGATGAGCGGCCAGGGCCACATGGAGACCGTGAAGAGCGCCAAGCGG CTGGACGAGGGCGTGAGCGTGCTGCGGGTGCCCCTGACCCAGCTGAAGCTG AAGGACCTGGAGAAGATGGTGAACCGGGAGCGGGAGCCCAAGCTGTACGAG GCCCTGAAGGCCCGGCTGGAGGCCCACAAGGACGACCCCGCCAAGGCCTTC GCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGGACCCAGCAGGTG AAGGCCGTGCGGGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGTGCGGAA CCACAACGGCATCGCCGACAACGCCACCATGGTGCGGGTGGACGTGTTCGA GAAGGGCGACAAGTACTACCTGGTGCCCATCTACAGCTGGCAGGTGGCCAAG GGCATCCTGCCCGACCGGGCCGTGGTGCAGGGCAAGGACGAGGAGGACTG GCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGCCTGCACCCCAACGAC CTGGTGGAGGTGATCACCAAGAAGGCCCGGATGTTCGGCTACTTCGCCAGCT
Description Sequence SEQ ID No. GCCACCGGGGCACCGGCAACATCAACATCCGGATCCACGACCTGGACCACA AGATCGGCAAGAACGGCATCCTGGAGGGCATCGGCGTGAAGACCGCCCTGA GCTTCCAGAAGTACCAGATCGACGAGCTGGGCAAGGAGATCCGGCCCTGCC GGCTGAAGAAGCGGCCCCCCGTGCGGAGCGGCAAGCGGACCGCCGACGGC
AGCGAGTTCGAGAGCCCCAAGAAGAAGCGGAAGGTGGAG GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG open reading frame for CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS1 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA
ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA 165
Description Sequence SEQ ID No. AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT
Description Sequence SEQ ID No. ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCTGGC
AGCAAAGAGAAGCAGAACAACA open reading frame of GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG to cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS2 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA
ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG 166
Description Sequence SEQ ID No. ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA
Description Sequence SEQ ID No. AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCAGGC
AGCAAAGAGAAGCAGAACAACA open reading frame of GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG to cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS3 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA
AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAG 167
Description Sequence SEQ ID No. GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT
Description Sequence SEQ ID No. CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGGC
ACCGGCAAAGAGAGAAAGAACAACA open reading frame of GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG to cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS4 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG
CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG 168
Description Sequence SEQ ID No. AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA
Description Sequence SEQ ID No. ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCAGGC
AGCAAAGAGACCGAGAACAACA GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG open reading frame for CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS5 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG 169
Description Sequence SEQ ID No. Sequence of AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC coding of TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA proteins of CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCGGAGTCGACGCAAGCG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA
Description Sequence SEQ ID No. TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCAGAGC
AGCAAAGAGACCGAGAACAACA Table of GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG open read CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG 170
Description Sequence SEQ ID NO cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC to NLS6 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC
Description Sequence SEQ ID No. TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC
Description Sequence SEQ ID No. AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCGCAGC
AGCAAAGAGAAGCTGGAGCATGGCAGCA open reading frame of GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG to cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS7 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG
AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC 171
Description Sequence SEQ ID No. GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG
Description Sequence SEQ ID No. AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCGCAGC
AGCAAAGAGAGTCTGGAGCATGGCATTC GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG open reading frame for CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS8 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC
AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA 172
Description Sequence SEQ ID No. GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG
Description Sequence SEQ ID No. AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCGCAGC
AGCAAAGAGAAGCTGGAGCATGGCATTC GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG open reading frame for CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS9 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA
CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG 173
Description Sequence SEQ ID No. GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC
Description Sequence SEQ ID No. TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCGCAGC
AGCAAAGAGAAAGTACTTCGCAGCA open reading frame of GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG to cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS10 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG sequence encoding AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion protein) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC
ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAAGAGCAACTTCGACCTGGCAGAAGACG 174
Description Sequence SEQ ID No. CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG
Description Sequence SEQ ID No. AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCAGAGC
AGCAAAGAGAAAGGCATTCGCAGCA GACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG open reading frame for CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGG cas9 with AAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTC NLS12 (no GACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA start codon or stop GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC; GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCC TGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGT suitable for inclusion in the sequence 175 CGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC
Description Sequence SEQ ID No. coding of TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAA proteins of CCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATAC fusion) AACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGG CAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGC ACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTG AGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACG CAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCT GGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTG AGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAA AGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGA CCTGACACTGCTGAAGGCACTGGTCAGACCAGCAGCTGCCGGAAAAGTACAAG GAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATG GACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAA AGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGA ACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAA GAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCG CACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAA AGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTC GACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAA CATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGA AGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAG ACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGA CAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTG AGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCC TGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATC CACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCG GACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGG CAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAA GGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAA AACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAA TCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGT
Description Sequence SEQ ID No. CGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCCAGAAC GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACT ACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGA CAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTC CCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGA ACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAG AGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTC GAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGA ACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACA CTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGG TCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGT CGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAAC AGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCT GATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAG AAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTAC GGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAG GTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAA TCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAA GCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGT ACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTC CTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACG AACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTG GACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGC AGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAA AGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGA AACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCAGAGC
AGCAAAGAGAAAGTACTTCGCAGTC Transcript of GGGAAGCUCAGAAUAAACGCUCAACUUUGGCCGGAUCUGCCACCAUGGACA mRNA with XBG AGAAGUACAGCAUCGGCCUGGACAUCGGCACCAACAGCGUGGGCUGGGCC UTRs and ORF of GUGAUCACCGACGAGUACAAGGUGCCCAGCAAGAAGUUCAAGGUGCUGGG 176
Description Sequence SEQ ID No. Cas9 with CAACACCGACAGACACAGCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUU U codons CGACAGCGGCGAGACCGCCGAGGCCACCAGACUGAAGAGAACCGCCAGAAGAGAGAGAACCGCCAGAAAGAACAGAAUCUGCUACCUGCAGGAGAUCUUCAG Table 4 CAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACAGAGA Table 4 CAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACAGAGA Table 4 CAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACAGAGA Table 4 CAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACAGAGA GCUUCCUGGUGGAGGAGGACAAGAAGCACGAGAGACACCCCAUCUUCGGC AACAUCGUGGACGAGGUGGCCUACACGAGAAGUACCCCACCAUCUACCAC CUGAGAAAGAAGCUGGUGGACAGCACCGACAAGGCCGACCUGAGACUGAUC UACCUGGCCCUGGCCCACAUGAUCAAGUUCAGAGGCCACUUCCUGAUCGAG GGCGACCUGAACCCCGACAACAGCGACGUGGACAAGCUGUUCAUCCAGCUG GUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCAGCGGC GUGGACGCCAAGGCCAUCCUGAGCGCCAGACUGAGCAAGAGCAGAAGACU GGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCG GCAACCUGAUCGCCCUGAGCCUGGGCCUGACCCCCAACUUCAAGAGCAACU UCGACCUGGCCGAGGACGCCAAGCUGCAGCUGAGCAAGGACACCUACGAC GACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCU GUUCCUGGCCGCCAAGAACCUGAGCGACGCCAUCCUGCUGAGCGACAUCC UGAGAGUGAACACCGAGAUCACCAAGGCCCCCCUGAGCGCCAGCAUGAUCA AGAGAUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGA GACAGCAGCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGAGCAAGA ACGGCUACGCCGGCUACAUCGACGGCGGCGCCAGCCAGGAGGAGUUCUAC AAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGCUGCU GGUGAAGCUGAACAGAGAGGACCUGCUGAGAAAGCAGAGAACCUUCGACAA CGGCAGCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGA GAAGACAGGAGGACUUCUACCCCUUCCUGAAGGACAACAGAGAGAAGAUCG AGAAGAUCCUGACCUUCAGAAUCCCCUACUACGUGGGCCCCCUGGCCAGAG GCAACAGCAGAUUCGCCUGGAUGACCAGAAAGAGCGAGGAGACCAUCACCC CCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCAGCGCCCAGAGCUUC AUCGAGAGAAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUG CCCAAGCACAGCCUGCUGUACGAGUACUUCACCGUGUACAACGAGCUGACC AAGGUGAAGUACGUGACCGAGGGCAUGAGAAAGCCCGCCUUCCUGAGCGG CGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAAACAGAAAGGU GACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAGUGCUUCGA CAGCGUGGAGAUCAGCGGCGUGGAGGACAGAUUCAACGCCAGCCUGGGCA CCUACCACGACCUGCUGAAGAUCAAUCAAGGACAAGGACUUCCUGGACAACG AGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUC GAGGACAGAGAGAUGAUCGAGGAGAGACUGAAGACCUACGCCCACCUGUUC GACGACAAGGUGAUGAAGCAGCUGAAGAGAAGAAGAUACACCGGCUGGGGC
Description Sequence SEQ ID No. AGACUGAGCAGAAAGCUGAUCAACGGCAUCAGAGACAAGCAGAGCGGCAAG ACCAUCCUGGACUUCCUGAAGAGCGACGGCUUCGCCAACAGAAACUUCAUG CAGCUGAUCCACGACGACAGCCUGACCUUCAAGGAGGACAUCCAGAAGGCC CAGGUGAGCGGCCAGGGCGACAGCCUGCACGAGCACAUCGCCAACCUGGC CGGCAGCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGG ACGAGCUGGUGAAGGUGAUGGGCAGACACAAGCCCGAGAACAUCGUGAUC GAGAUGGCCAGAGAGAACCAGACCACCAGAAGGGCCAGAAGAACAGCAGA GAGAGAAUGAAGAGAAUCGAGGAGGGCAUCAAGGAGCUGGGCAGCCAGAU CCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUA CCUGUACUACCUGCAGAACGGCAGAGACAUGUACGUGGACCAGGAGCUGG ACAUCAACAGACUGAGCGACUACGACGUGGACCACAUCGUGCCCCAGAGCU UCCUGAAGGACGACAGCAUCGACAACAAGGUGCUGACCAGAAGCGACAAGA ACAGAGGCAAGAGCGACAACGUGCCCAGCGAGGAGGUGGUGAAGAAGAUG AAGAACUACUGGAGACAGCUGCUGAACGCCAAGCUGAUCACCCAGAGAAAG UUCGACAACCUGACCAAGGCCGAGAGAGGCGGCCUGAGCGAGCUGGACAA GGCCGGCUUCAUCAAGAGACAGCUGGUGGAGACCAGACAGAUCACCAAGCA CGUGGCCCAGAUCCUGGACAGCAGAAUGAACACCAAGUACGACGAGAACGA CAAGCUGAUCAGAGAGGUGAAGGUGAUCACCCUGAAGAGCAAGCUGGUGA GCGACUUCAGAAAGGACUUCCAGUUCUACAAGGUGAGAGAGAUCAACAACU ACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUGGGCACCGCCCUG AUCAAGAAGUACCCCAAGCUGGAGAGCGAGUUCGUGUACGGCGACUACAAG GUGUACGACGUGAGAAAGAUGAUCGCCAAGAGCGAGCAGGAGAUCGGCAA GGCCACCGCCAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAGAC CGAGAUCACCCUGGCCAACGGCGAGAUCAGAAAGAGACCCCUGAUCGAGAC CAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGGCAGAGACUUCGCCA CCGUGAGAAAGGUGCUGAGCAUGCCCCAGGUGAACAUCGUGAAGAAGACC GAGGUGCAGACCGGCGGCUUCAGCAAGGAGAGCAUCCUGCCCAAGAGAAA CAGCGACAAGCUGAUCGCCAGAAAGAAGGACUGGGACCCCAAGAAGUACGG CGGCUUCGACAGCCCCACCGUGGCCUACAGGCGUGCUGGUGGUGGCCAAGG UGGAGAAGGGCAAGAGCAAGAAGCUGAAGAGCGUGAAGGAGCUGCUGGGC AUCACCAUCAUGGAGAGAAGCAGCUUCGAGAAGAACCCCAUCGACUUCCUG GAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCC AAGUACAGCCUGUUCGAGCUGGAGAACGGCAGAAAGAGAAUGCUGGCCAGC GCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCAGCAAGUACGU GAACUUCCUGUACCUGGCCAGCCACUACGAGAAGCUGAAGGGCAGCCCCGA GGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGA CGAGAUCAUCGAGCAGAUCAGCGAGUUCAGCAAGAGAGUGAUCCUGGCCGA
Description Sequence SEQ ID No. CGCCAACCUGGACAAGGUGCUGAGCGCCUACAACAAGCACAGAGACAAGCC CAUCAGAGAGCAGGCCGAGAACAUCAUCCACCUGUUCACCCUGACCAACCU GGGCGCCCCCGCCGCCUUCAAAGUACUUCGACACCACCAUCGACAGAAAGAG AUACACCAGCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGAGCAU CACCGGCCUGUACGAGAGACCAGAAUCGACCUGAGCCAGCUGGGCGGCGACG GCGGCGGCAGCCCCAAGAAGAAGAGAAAGGUGUGACUAGCACCAGCCUCAA GAACACCCGAAUGGAGUCUCUAAGCUACAUAAAUACCACUUACACUUUACAA AAUGUUGUCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAAAGAAA GOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAUCUAG GGGAAGCUCAGAAUAAACGCUCAACUUUGGCCGGAUCUGCCACCAUGGACA mRNA transcript with XBG AGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCC UTRs and ORF GUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGG cas9 with codons CAACACCGACCGGCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUU The low CGACUCCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGC 1 GGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUC Table 4 UCCAACGAGAUGGCCAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGA GUCCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCCAUCUUCG GCAACAUCGUGGACGAGGUGGCCUACACGAGAAGUACCCCACCAUCUACC ACCUGCGGAAGAAGCUGGUGGACUCCCACCGACAAGGCCGACCUGCGGCUG AUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUC GAGGGCGACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUCAUCCAG CUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCUCC GGCGUGGACGCCAAGGCCAUCCUGUCCGCCCGGCUGUCCAAGUCCCGGCG GCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGU UCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCA ACUUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUAC GACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGA CCUGUUCCUGGCCGCCAAGAACCUGUCCGACGCCAUCCUGCUGUCCGACA UCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGA UCAAGCGGUACGAGCGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUG GUGCGGCAGCAGCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGUC CAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCUCCCAGGAGGAGU UCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGC
UGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUC GACAACGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUC 177
Description Sequence SEQ ID No. CUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGGGAGAA GAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGG CCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACC AUCACCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCUCCGCCCA GUCCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAA GGUGCUGCCCAAGCACUCCCUGCUGUACGAGUACUUCACCGUGUACAACGA GCUGACCAAGGUGAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCCUUCC UGUCCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAAGACCAAC CGGAAGGUGACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAG UGCUUCGACUCCGUGGAGAUCUCCGGGCUGGAGGACCGGUUCAACGCCUC CCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCU GGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGA CCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCC CACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACAC CGGCUGGGGCCGGCUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGC AGUCCGGCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUUCGCCAACC GGAACUUCAUGCAGCUGAUCCACGACGACUCCCUGACCUUCAAGGAGGACA UCCAGAAGGCCCAGGUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUC GCCAACCUGGCCGGCUCCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGU GAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGA ACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGA AGAACUCCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUG GGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAAC GAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGGACAUGUACGUGGA CCAGGAGCUGGACAUCAACCGGCUGUCCGACUACGACGUGGACCACAUCG UGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCUGACCC GGUCCGACAAGAACCGGGGCAAGUCCGACAACGUGCCCUCCGAGGAGGUG GUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUC ACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGUC CGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGC AGAUCACCAAGCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGU ACGACGAGAACGACAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGU CCAAGCUGGUGUCCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGG GAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUG GGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGUCCGAGUUCGUGUA CGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGUCCGAGC AGGAGAUCGGCAAGGCCACCGCCAAGUACUUCUUCUACUCCAACAUCAUGA
Description Sequence SEQ ID No. ACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGC CCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUUGUGGGACAAGGGC CGGGACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGGUGAACAU CGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCAUCC UGCCCAAGCGGAACUCCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGAC CCCAAGAAGUACGGCGGCUUCGACUCCCCCACCGUGGCCUACUCCGUGCU GGUGGUGGCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGA AGGAGCUGCUGGGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAAC CCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCU GAUCAUCAAGCUGCCCAAGUACUCCCUGUUCGAGCUGGAGAACGGCCGGAA GCGGAUGCUGGCCUCCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCC UGCCCUCCAAGUACGUGAACUUCCUGUACCUGGCCUCCCACUACGAGAAGC UGAAGGGCUCCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAG CACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCUCCGAGUUCUCCAAG CGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGUCCGCCUACAA CAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCU GUUCACCCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACAC CACCAUCGACCGGAAGCGGUACACCUCCACCAAGGAGGUGCUGGACGCCAC CCUGAUCCACCAGUCCAUCACCGGCCUGUACGAGAGACCCGGAUCGACCUGUC CCAGCUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGU GACUAGCACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAU ACCAACUUACACUUUACAAAAUGUUGUCCCCAAAAUGUAGCCAUUCGUAUC UGCUCCUAAUAAAAAGAAAGUUUCUCACAUUCUCUCGAGAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG GGGAAGCUCAGAAUAAACGCUCAACUUUGGCCGGAUCUGCCACCAUGGACA mRNA transcript with XBG AGAAGUACAGCAUCGGCCUGGACAUCGGCACCAACAGCGUGGGCUGGGCC UTRs and ORF GUGAUCACCGACGAGUACAAGGUGCCCAGCAAGAAGUUCAAGGUGCUGGG cas9 with codons CAACACCGACCGGCACAGCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUU U / A 1 Low CGACAGCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGC GGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUC Table 4 AGCAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACCGGCUGGAGGA GAGCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCCAUCUUCG GCAACAUCGUGGACGAGGUGGCCUACACGAGAAGUACCCCACCAUCUACC ACCUGCGGAAGAAGCUGGUGGACAGCACCGACAAGGCCGACCUGCGGCUG
AUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUC GAGGGCGACCUGAACCCCGACAACAGCGACGUGGACAAGCUGUUCAUCCAG 178
Description Sequence SEQ ID No. CUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCAGC GGCGUGGACGCCAAGGCCAUCCUGAGCGCCCGGCUGAGCAAGAGCCGGCG GCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGU UCGGCAACCUGAUCGCCCUGAGCCUGGGCCUGACCCCCAACUUCAAAGAGCA ACUUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGAGCAAGGACACCUAC GACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGA CCUGUUCCUGGCCGCCAAGAACCUGAGCGACGCCAUCCUGCUGAGCGACA UCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGAGCGCCAGCAUG AUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUG GUGCGGCAGCAGCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGAG CAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCAGCCAGGAGGAGU UCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGC UGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUC GACAACGGCAGCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUC CUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGGGAGAA GAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGG CCCGGGGCAACAGCCGGUUCGCCUGGAUGACCCGGAAGAGCGAGGAGACC AUCACCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCAGCGCCCA GAGCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAA GGUGCUGCCCAAGCACAGCCUGCUGUACGAGUACUUCACCGUGUACAACGA GCUGACCAAGGUGAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCCUUCC UGAGCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAAC CGGAAGGUGACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAG UGCUUCGACAGCGUGGAGAUCAGCGGGCUGGAGGACCGGUUCAACGCCAG CCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCU GGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGA CCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCC CACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACAC CGGCUGGGGCCGGCUGAGCCGGAAGCUGAUCAACGGCAUCCGGGACAAGC AGAGCGGCAAGACCAUCCUGGACUUCCUGAAGAGCGACGGCUUCGCCAACC GGAACUUCAUGCAGCUGAUCCACGACGACAGCCUGACCUCUCAAGGAGGACA UCCAGAAGGCCCAGGUGAGCGGCCAGGGCGACAGCCUGCACGAGCACAUC GCCAACCUGGCCGGCAGCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGU GAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGA ACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCCAGAAGGGCCAGA AGAACAGCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUG GGCCAGCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAAC
Description Sequence SEQ ID No. GAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGGACAUGUACGUGGA CCAGGAGCUGGACAUCAACCGGCUGAGCGACUACGACGUGGACCACAUCG UGCCCCAGAGCUUCCUGAAGGACGACAGCAUCGACAACAAGGUGCUGACCC GGAGCGACAAGAACCGGGGCAAGAGCGACAACGUGCCCAGCGAGGAGGUG GUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUC ACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGAG CGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGC AGAUCACCAAGCACGUGGCCCAGAUCCUGGACAGCCGGAUGAACACCAAGU ACGACGAGAACGACAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGA GCAAGCUGGUGAGCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGG GAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUG GGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGAGCGAGUUCGUGUA CGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGAGCGAGC AGGAGAUCGGCAAGGCCACCGCCAAGUACUUCUUCUACAGCAACAUCAUGA ACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGC CCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUUGUGGGACAAGGGC CGGGACUUCGCCACCGUGCGGAAGGUGCUGAGCAUGCCCCAGGUGAACAU CGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCAGCAAGGAGAGCAUCC UGCCCAAGCGGAACAGCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGAC CCCAAGAAGUACGGCGGCUUCGACAGCCCCACCGUGGCCUACAGCGUGCU GGUGGUGGCCAAGGUGGAGAAGGGCAAGAGCAAGAAGCUGAAGAGCGUGA AGGAGCUGCUGGGCAUCACCAUCAUGGAGCGGAGCAGCUUCGAGAAGAAC CCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCU GAUCAUCAAGCUGCCCAAGUACAGCCUGUUCGAGCUGGAGAACGGCCGGAA GCGGAUGCUGGCCAGCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCC UGCCCAGCAAGUACGUGAACUUCCUGUACCUGGCCAGCCACUACGAGAAGC UGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAG CACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCAGCGAGUUCAGCAAG CGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGAGCGCCUACAA CAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCU GUUCACCCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACAC CACCAUCGACCGGAAGCGGUACACCAGCACCAAGGAGGUGCUGGACGCCAC CCUGAUCCACCAGAGCAUCACCGGCCUGUACGAGAGACCCGGAUCGACCUGAG CCAGCUGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGCGGAAGGUGU GACUAGCACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAU ACCAACUUACACUUUACAAAAUGUUGUCCCCAAAAUGUAGCCAUUCGUAUC UGCUCCUAAUAAAAAGAAAGUUUCUCACAUUCUCUCGAGAAAAAAAAAAAAAAA
Description Sequence SEQ ID No. AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAUCUAG GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTG mRNA transcript with CAGGCCTTATTCGGATCCGCCACCATGGACAAGAAGTACAGCATCGGACTGG ORF encoding ACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGT cas9 with CCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAG HiBit label AAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAA HSD 5'UTR and CAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAAT ALB 3'UTR human CTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGC TTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACG AAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAA GTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAG GCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAG GACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAA GCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGA TCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAA GAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAAGAAGAAC GGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCA AGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACAC ATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCA GACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACA TCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGAT CAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTC AGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGA ACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAA GTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTC AAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAA GCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACA GGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCC TGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAG ATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTC GAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGA CAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCA CAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAAT
CGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAG GAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAG 179
Description Sequence SEQ ID No. TCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGAT CATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAA GACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAG ACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAG AGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAA TCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGG ATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCA AGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACG AACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCA GACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCG GAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGAC AGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACT GGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAAC GAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACC AGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCC GCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGC GACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGA AGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAG AAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGAC AAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGC ACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGA CAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGC GACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCA CCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAG AAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACG ACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGC AAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACT GGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACA GGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCC TGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGG ATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCA AGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACA GTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGA AGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAG CTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTC AAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAA ACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACG
Description Sequence SEQ ID No. AACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTA CGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTC GAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATA CAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCAC CTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACA CAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAAC ACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGC CAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAAGGTCAGC GAAAGCGCAACACCGGAAAGCGTCAGCGGATGGAGACTGTTCAAGAAGATCA GCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGA AAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAA GCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCCTTTTCTC TGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAGAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAATCTAG GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATCGCCACC mRNA transcript with ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT ORF encoding GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCT cas9 with GGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTG HiBit label TTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAA CMV-1 5 'UTR and GAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC ALB 3'UTR human AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCT TCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACAT CGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGA AAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGG CACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCT GAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACA TACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAA AGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGAT CGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCA CTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAG ACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCT GCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA
CAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACGAACACCACCA GGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTAC 180
Description Sequence SEQ ID No. AAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACG GAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAA GATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTG AGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGG GAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAA GGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACG TCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAAGAG CGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCA AGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGA ACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTAC AACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCAT TCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAA CAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAAT GCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCT GGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGAC AACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTT CGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTC GACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAA GACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGAC AATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGT CAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTG GTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAA GAGAAAACCAGACAAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAA GAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACAC CCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGC AGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAG CGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGC ATCCAAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACA ACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCA GAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAG CTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCA GAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGT CATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCT ACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGACGCATACCTGAA
Description Sequence SEQ ID No. CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA GCCCGAAGAAGAAGAGAAAGGTCAGCGAAAGCGCAACACCGGAAAGCGTCA GCGGATGGAGACTGTTCAAGAAGATCAGCTAGCTAGCCATCACATTTAAAAGC ATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTC ATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAA TTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAA GAACCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
The transcribed mRNA with GGGAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGCGCCA CCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGG ORF encoding ATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTC cas9 with CTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGC HiBit label TGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAG CMV-2 5 'UTR and AAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCA ALB 3' UTR 181 GCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAG
Description Sequence SEQ ID No. human CTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAAC ATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGA GAAAGAAGCTGGTCGACAGCCACAGACAAGGCAGACCTGAGACTGATCTACCT GGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGAC CTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGA CATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGC AAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTG ATCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCG CACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGA AGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAAC CTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGA ACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAAACACAGAAAT CACAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCAC CAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGT ACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGA CGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAA AAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGC TGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCT GGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTG AAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTA CGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAGAAAG AGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAG CAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCC GAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTC TACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGG CATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGAC AAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATC GAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCT GGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACA CTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACACCT GTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGG GGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGGCGACGGATTCGCAAACAGAAACTTCATG CAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCAC AGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAG GAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGA
Description Sequence SEQ ID No. ACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATG GCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAA TGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGA ACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTAC CTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGAC TGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGA CAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGC GACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGAC AGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAA GGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAG ACAGCTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGAC AGCAGAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCA AGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCA GTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACC TGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAG CGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAA AGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAA CATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAA AGAGACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAA GGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAAC ATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCC TGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCC GAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTC GTCGCAAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAAC TGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGA CTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAG CTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGG CAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGT ACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCC GGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTG GACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAG ACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCC GATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAAGATA CACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACA GGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGA GGAAGCCCGAAGAAGAAGAGAAAAGGTCAGCGAAAGCGCAACACCGGAAAGC
Description Sequence SEQ ID No. GTCAGCGGATGGAGACTGTTCAAGAAGATCAGCTAGCTAGCCATCACATTTAA AAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCT TATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAAC ATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATG GAAAGAACCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAA GGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGCGCCAC mRNA transcript with CATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGA ORF encoding TGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCC cas9 with TGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCT HiBit label GTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGA 3-CMV 5 'UTR and AGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAG ALB 3'UTR human CAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGC TTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACA TCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAG AAAGAAGCTGGTCGACAGCCACAGACAAGGCAGACCTGAGACTGATCTACCTG GCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACC TGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGAC ATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCA AAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGA TCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGC ACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAA GACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACC TGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAA CCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACC AGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGAAAAGTA CAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGAC GGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAA AGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCT GAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTG GGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGA AGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTAC GTCGGACCGCTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGA
GCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGC AAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCG 182
Description Sequence SEQ ID No. AACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTA CAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCA TTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAA ACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGA ATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGC CTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGG ACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACT GTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGT TCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGG AAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAG ACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCA GCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAG GTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGA AGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGC AAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATG AAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAAC ACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTG AGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACA GCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGA CAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAG CTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGG CAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACA GCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGC AGAATGAACACAAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGG TCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTC TACAAGGTCAGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAA CGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAA TTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGA GCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAG ACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAAATCGTCTGGGACAAGGGA AGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCG TCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCC GAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAA GAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTC
Description Sequence SEQ ID No. GCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGC TGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTC CTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGC CGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAG CGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGT CAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAA GACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACG AAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGC AAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATC AGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAG CACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGAC TGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAA GCCCGAAGAAGAAGAGAAAGGTCAGCGAAAGCGCAACACCGGAAAGCGTCA GCGGATGGAGACTGTTCAAGAAGATCAGCTAGCTAGCCATCACATTTAAAAGC ATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTC ATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAA TTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAA GAACCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
The transcribed mRNA with GGGCATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGGTCCCCACAG ACTCAGAGAGAACCCACCCGCCACCATGGACAAGAAGTACAGCATCGGACTG ORF encoding GACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGG cas9 with TCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAA HiBit label GAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCA HBA 5 'UTR and ACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAAT ALB 3'UTR human CTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGC TTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACG AAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAA GTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAG GCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAG GACACTTCCTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAA GCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGA TCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAA
GAGCAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAAAAGAAGAAC GGACTGTTCGGAAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCA 183
Description Sequence SEQ ID No. AGAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACAC ATACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCA GACCTGTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACA TCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGAT CAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTC AGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGA ACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAA GTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTC AAGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAA GCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACA GGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCC TGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAG ATTCGCATGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTC GAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGA CAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCA CAGAAGGAATGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAAT CGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAG GAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAG TCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGAT CATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAA GACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAG ACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAG AGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAA TCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGG ATTCGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCA AGGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACG AACACATCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCA GACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCG GAAAACATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGAC AGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACT GGGAAGCCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAAC GAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACC AGGAACTGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGTCCC GCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGC GACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGA AGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAG
Description Sequence SEQ ID No. AAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGAC AAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAGC ACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGA CAAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGC GACTTCAGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCA CCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAG AAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACG ACGTCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGC AAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACT GGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAACA GGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCC TGAGCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGG ATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCA AGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACA GTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGA AGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAG CTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTC AAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAA ACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACG AACTGGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTA CGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTC GAACAGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATA CAACAAGCACAGAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCAC CTGTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACA CAACAATCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAAC ACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGC CAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAAGGTCAGC GAAAGCGCAACACCGGAAAGCGTCAGCGGATGGAGACTGTTCAAGAAGATCA GCTAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGA AAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAA GCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCCTTTTCTC TGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAGAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAA transcript from GGGACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC mRNA with ORF GGATCTCGCCACCATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACA 184
Description Sequence SEQ ID No. encoding AACAGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGA cas9 with AGTTCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCTGAT HiBit label CGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTGAAG HBB 5 'UTR and AGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCA ALB 3'UTR human GGAAATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGA CTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACCCGA TCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACAATC TACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGAC TGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTGATC GAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCAGC TGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGG AGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTG GAAAACCTGATCGCACAGCTGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAA ACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGA CCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGA CCTGGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCTGTTCCTG GCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCA ACACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAAGAGATACGA CGAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTG CCGGAAAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATACGCAG GATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCC GATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGA GAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCCCGCACC AGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTTCTA CCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAA TCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGAT GACAAGAAAGAGCGAAGAAAAAATCACACCGTGGAACTTCGAAGAAGTCGTC GACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACA AGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATA CTTCACAGTCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGA GAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCT GTTCAAGACAAAACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTCA AGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTCGAAGACAGATT CAACGCAAGCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAAG GACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGA CACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATAC GCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAAGAAGATACA
Description Sequence SEQ ID No. CAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCA GAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAGA AACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCA GAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAA CCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTC GTCGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAG AGAAAGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATC CTGAAGGAACACCCGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACC TGTACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTG AAGGACGACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAACAGAG GAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTA CTGGAGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAAC CTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGGCAGGATTC ATCAAGAGACAGCTGGTCGAAAACAAGACAGATCACAAAGCACGTCGCACAGA TCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACAAGCTGATCAG AGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAG GACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACACACGA CGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAG CTGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGA TGATCGCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTT CTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAG AAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGAAAACAGGAGAAATCGT CTGGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCG CAGGTCAACATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGG AAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAAGGA CTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGACAGTCGCATACAG CGTCCTGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGAAGAGC GTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGA ACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCT GATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAG AGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCACTG CCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGA AGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAA GCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTC ATCCTGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACA
Description Sequence SEQ ID No. GAGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACT GACAAACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGAC AGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGATCCACC AGAGCATCACAGGACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGG AGACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAAGGTCAGCGAAAGCGCAAC ACCGGAAAGCGTCAGCGGATGGAGACTGTTCAAGAAGATCAGCTAGCTAGCC ATCACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAA GATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCT GTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAAT TAATAAAAAATGGAAAGAACCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA GGGAAGCTCAGAATAAACGCTCAACTTTGGCCGGATCTCGCCACCATGGACA mRNA transcript with AGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGCAGT ORF encoding CATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAAC cas9 with ACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACA HiBit label GCGGAGAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATA XBG 5 'UTR and CACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAA ALB 3'UTR human TGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGT CGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGAAACATCGTCGAC GAAGTCGCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCT GGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGGCA CACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGG ACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGACATACAACCA GCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATC CTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGC TGCCGGGAGAAAAAGAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCT GGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGACGCAAAG CTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGCAC AGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGA CGCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCA CCGCTGAGCGCAAGCATGATCAAAGATACGACGAACACCACCAGGACCTGA CACTGCTGAAGGCACTGGTCAGACGCAGCTGCCGGAAAAGTACAAGGAAAT CTTCTTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCA AGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGATGGACG
GAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAAGCA GAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCACCTGGGAGAACTG 185
Description Sequence SEQ ID No. CACGCAATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACA GAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGGACCG CTGGCAAGAGGAAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGAAA CAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACA GAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAAAG GTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAACT GACAAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGC GGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGG TCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGAC AGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACAT ACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGA AAACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACA GAGAAATGATCGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGC AGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGG ACTTCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGCTGATCCAC GACGACAGCCTGACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGAC AGGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGT CATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAGAAAAC CAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCG AAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGA AGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACG ACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAA CAAGGTCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCG AGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACG CAAAGCTGATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGG AGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAA ACAAGACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACA CAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCATCACACT GAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTC AGAGAAATCAACAACTACCACCACGCACACGACGCATACCTGAACGCAGTCG TCGGAACAGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTA CGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGAACAG GAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTT CTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGAGACCGCTG
Description Sequence SEQ ID No. ATCGAAACAAACGGAGAAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACT TCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGAA GACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGA AACAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACG GAGGATTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGT CGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATC ACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAG CAAAGGGATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTA CAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGA GAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCC TGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGGAAGACAACGA ACAGAAGCAGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCATC GAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGG ACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACA GGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGCACCGGCA GCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAA GGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTACGAA ACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG AAGAAGAGAAAGGTCAGCGAAAGCGCAACACCGGAAAGCGTCAGCGGATGG AGACTGTTCAAGAAGATCAGCTAGCTAGCCATCACATTTAAAAGCATCTCAGC CTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTT TTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTA ATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTC GGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS1 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK
EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL 186
Description Sequence SEQ ID No. DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSLAAKRSRTT MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS2 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH
YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE 187
Description Sequence SEQ ID No.
TRIDLSQLGGDGGGSQAAKRSRTT MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS3 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGDGGGSPAPAKRERTT 188 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS4 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP
KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT 189
Description Sequence SEQ ID No. LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSQAAKRPRTT MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS5 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR 190
Description Sequence SEQ ID No. DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSRAAKRPRTT MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS6 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGDGGGSAAAKRSWSMAA 191 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS7 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL
ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK 192
Description Sequence SEQ ID No. EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSAAAKRVWSMAF MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS8 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK
KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 193
Description Sequence SEQ ID No. YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSAAAKRSWSMAF MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS9 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR
DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE TRIDLSQLGGDGGGSAAAKRKYFAA 194 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS10 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED
LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP 195
Description Sequence SEQ ID No. KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSRAAKRKAFAA MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE amino acid sequence TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH to cas9 with ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE NLS11 GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW
DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK 196
Description Sequence SEQ ID No. GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE
TRIDLSQLGGDGGGSRAAKRKYFAV RNA guide G506 mA * mU * mA * CCAGUCCAGCGAGGCAGGUUUUAGAmGmCmUmAmGmAmAmA directed to mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA TTR mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU * mU * mU * mU 197 RNA guide G510 mA * mC * mU * UGUCUUCUCUAUACCCAGUUUUAGAmGmCmUmAmGmAmAmAm directed to UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm TTR AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU * mU * m U * mU 198 * = Connection PS ; 'm' = nucleotide 2'-O-Me

权利要求:
Claims (1)
[1]
1. Messenger ribonucleic acid (mRNA), characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has a uridine content that varies from its minimum content of uridine at 150% of the minimum uridine content.
2. Messenger ribonucleic acid (mRNA), characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has a uridine dinucleotide content that varies from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.
3. Messenger ribonucleic acid (mRNA), characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has an adenine content that varies from its minimum content of uridine at 150% of the minimum adenine content.
4. Messenger ribonucleic acid (mRNA), characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has an adenine dinucleotide content that varies from its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content.
5. Messenger ribonucleic acid (mRNA), characterized in that it comprises a sequence with at least 90% identity to any of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175, wherein the mRNA comprises an open reading frame encoding a binding to DNA guided by RNA.
6. Messenger ribonucleic acid (mRNA), characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, wherein the open reading frame has at least 90% identity with any of the SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175 for at least its first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides.
7. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the open reading frame consists of a set of codons of which at least 75% of the codons are (i) codons listed in Table 1, Table 2 or Table 3, or (ii) a set of codons listed in Table
4.
8. Messenger ribonucleic acid (mRNA) encoding an RNA-guided DNA binding agent, characterized in that it comprises an open reading frame encoding an RNA-guided DNA binding agent, in which the open reading frame consists of a set of codons of which at least 75% of the codons are codons listed in Table 1, Table 2, Table 3 or (ii) a set of codons listed in Table 4.
9. Messenger ribonucleic acid (mRNA), according to claim 7 or 8, characterized in that the open reading frame consists of a set of codons of which at least 75% of the codons are codons of the low defined U 1 set in Table 4.
10. Messenger ribonucleic acid (mRNA), according to claim 7 or 8, characterized in that the open reading frame consists of a set of codons of which at least 75% of the codons are codons from the set A below defined in Table 4.
11. Messenger ribonucleic acid (mRNA), according to claim 7 or 8, characterized in that the open reading frame consists of a set of codons of which at least 75%
of the codons are codons from the low A/U set defined in Table 4.
12. Messenger ribonucleic acid (mRNA), according to claim 7 or 8, characterized in that the open reading frame consists of a set of codons of which at least 75% of the codons are half-life set codons long defined in Table 4.
13. Messenger ribonucleic acid (mRNA), according to any one of claims 7 to 12, characterized in that at least 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are (i) codons listed in Table 1, Table 2 or Table 3 or (ii) a set of codons listed in Table 4.
14. Messenger ribonucleic acid (mRNA) according to any one of claims 1 to 5 or 7 to 13, characterized in that the open reading frame has at least 90% identity with any of SEQ ID NO: 1 , 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175 for at least its first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides.
15. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the open reading frame has at least 90% identity with any of SEQ ID NOs: 1, 4, 7, 9 , 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175 for at least the first 10 %, 12%, 15%, 20%, 25%, 30% or 35% of your sequence.
16. Messenger ribonucleic acid (mRNA), according to any one of claims 1 to 4 or 6 to 15, characterized in that the mRNA comprises a sequence with at least 90% identity with any of SEQ ID NO: 1 or 107-
175.
17. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the open reading frame has a uridine dinucleotide content that varies from its minimum uridine dinucleotide content to 101%, 102 %, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum content of uridine dinucleotide.
18. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the open reading frame has a uridine content that varies from its minimum uridine content to 101%, 102%, 103% , 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum uridine content.
19. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the open reading frame has an adenine content that varies from its minimum uridine content to 101%, 102%, 103% , 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum adenine content.
20. Messenger ribonucleic acid (mRNA), according to any of the preceding claims, characterized in that the open reading frame has an adenine dinucleotide content that varies from its minimum adenine dinucleotide content to 101%, 102% , 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the minimum adenine dinucleotide content.
21. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that it comprises a 5' UTR with at least 90% identity with any of SEQ ID NOs: 32, 34, 36, 38 , 41 or 75-77.
22. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that a 3' UTR with at least 90% identity with any of SEQ ID NOs: 33, 35, 37, 39 or 40.
23. Messenger ribonucleic acid (mRNA), according to claim 21 or 22, characterized in that the mRNA comprises a 5' UTR and a 3' UTR from the same source.
24. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that it comprises a 5' capping selected from Cap0, Cap1 and Cap2.
25. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the open reading frame has codons that increase the translation of mRNA in a mammal.
26. Messenger ribonucleic acid (mRNA), according to claim 25, characterized in that the open reading frame has codons that increase the translation of mRNA in a specific organ of a mammal.
27. Messenger ribonucleic acid (mRNA), according to claim 26, characterized in that the organ is liver.
28. Messenger ribonucleic acid (mRNA), according to any one of claims 25 to 27, characterized in that the mammal is a human being.
29. Messenger ribonucleic acid (mRNA) according to any one of claims 25 to 28, characterized in that the codons increase the translation of the mRNA in the mammal in relation to the translation of an mRNA comprising an ORF with a sequence consisting of the SEQ ID NO: 5.
30. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that when mRNA is administered to a mammal in a pharmaceutical composition, the mammal exhibits a cytokine response at least 5 times lower than a mammal that has been administered an mRNA comprising an ORF encoding a Cas9 nuclease with greater than 150% of the minimum uridine content.
31. Messenger ribonucleic acid (mRNA), according to claim 30, characterized in that the mRNA comprising the ORF encoding Cas9 nuclease with more than 150% of the minimum uridine content has a sequence consisting of SEQ ID NO: 5.
32. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the RNA-guided DNA binding agent has double-stranded endonuclease activity.
33. Messenger ribonucleic acid (mRNA), according to claim 32, characterized in that the RNA-guided DNA binding agent comprises a Cas cleavase.
34. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the RNA-guided DNA binding agent has nickase activity.
35. Messenger ribonucleic acid (mRNA), according to claim 34, characterized in that the RNA-guided DNA binding agent comprises a Cas nickase.
36. Messenger ribonucleic acid (mRNA), according to any one of claims 1 to 31, characterized in that the RNA-guided DNA binding agent comprises a dCas DNA binding domain.
37. Messenger ribonucleic acid (mRNA) according to any one of claims 33 or 35 and 36, characterized in that the Cas cleavase, Cas nickase, or dCas DNA binding domain is a Cas9 cleavase binding domain, Cas9 nickase, or dCas9 DNA.
38. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the encoded RNA-guided DNA binding agent comprises a nuclear localization signal (NLS).
39. Messenger ribonucleic acid (mRNA), according to claim 38, characterized in that the NLS is linked to the C-terminus of the RNA-guided DNA binding agent.
40. Messenger ribonucleic acid (mRNA), according to claim 38, characterized in that the NLS is linked to the N-terminus of the RNA-guided DNA binding agent.
41. Messenger ribonucleic acid (mRNA), according to any one of claims 38 to 40, characterized in that the NLS comprises a sequence with at least 80%, 85%, 90% or 95% identity with any of the SEQ ID NOs: 78-91.
42. Messenger ribonucleic acid (mRNA), according to any one of claims 38 to 40, characterized in that the NLS comprises the sequence of any one of SEQ ID NOs: 78-
91.
43. Messenger ribonucleic acid (mRNA), according to any one of claims 38 to 42, characterized in that the NLS is encoded by a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to any of SEQ ID NOs: 92-104.
44. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 90% identity with SEQ ID NO: 4, 7 or 9.
45. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 95% identity with SEQ ID NO: 4, 7 or 9.
46. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 98% identity with SEQ ID NO: 4, 7 or 9.
47. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence 100% identical to SEQ ID NO: 4, 7 or 9.
48. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 90% identity with SEQ ID NO: 111, 114 or 117.
49. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 95% identity with SEQ ID NO: 111, 114 or 117.
50. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 98% identity with SEQ ID NO: 111, 114 or 117.
51. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence 100% identical to SEQ ID NO: 112, 122 or 125.
52. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 90% identity with SEQ ID NO: 112, 122 or 125.
53. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 95% identity with SEQ ID NO: 112, 122 or 125.
54. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 90% identity with SEQ ID NOs: 10, 11, 12, 14 , 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.
55. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 95% identity with SEQ ID NO: 10, 11, 12, 14 , 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66 or 107-175.
56. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence with at least 98% identity with SEQ ID NOs: 10, 11, 12, 14 , 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.
57. Messenger ribonucleic acid (mRNA), according to any one of claims 37 to 43, characterized in that the mRNA comprises a sequence 100% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.
58. Messenger ribonucleic acid (mRNA) according to any one of claims 37 to 57, characterized in that the mRNA encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 8 or 186-196.
59. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the RNA-guided DNA binding agent further comprises a heterologous functional domain.
60. Messenger ribonucleic acid (mRNA), according to claim 59, characterized in that the heterologous functional domain is a FokI nuclease.
61. Messenger ribonucleic acid (mRNA), according to claim 59, characterized by the fact that the heterologous functional domain is a transcriptional regulatory domain.
62. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that when an effective amount of the mRNA is administered to a mammal together with a guide RNA that directs the mammal's TTR gene in a composition pharmaceutical comprising lipid nanoparticles, an indel is formed at the TTR locus in at least 50% of the genomic DNA obtained from mammalian hepatocytes.
63. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that when an effective amount of the mRNA is administered to a mammal together with a guide RNA that directs the mammal's TTR gene in a composition pharmaceutical comprising lipid nanoparticles, the concentration of TTR in mammalian serum is reduced by at least 50%.
64. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that at least 10% of the uridine is replaced by a modified uridine.
65. Messenger ribonucleic acid (mRNA), according to claim 64, characterized in that the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine or 5-iodouridine.
66. Messenger ribonucleic acid (mRNA), according to claim 64, characterized in that the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.
67. Messenger ribonucleic acid (mRNA), according to claim 64, characterized in that the modified uridine is N1-methyl-pseudouridine.
68. Messenger ribonucleic acid (mRNA), according to claim 64, characterized in that the modified uridine is 5-methoxyuridine.
69. Messenger ribonucleic acid (mRNA), according to any one of claims 64 to 68, characterized in that 15% to 45% of the uridine is replaced by the modified uridine.
70. Messenger ribonucleic acid (mRNA), according to any one of claims 64 to 68, characterized in that at least 20% or at least 30% of the uridine is replaced by the modified uridine.
71. Messenger ribonucleic acid (mRNA), according to claim 70, characterized in that at least 80% or at least 90% of the uridine is replaced by the modified uridine.
72. Messenger ribonucleic acid (mRNA), according to claim 70, characterized in that 100% of uridine is replaced by modified uridine.
73. Messenger ribonucleic acid (mRNA) according to any one of claims 64 to 72, characterized in that when an effective amount of the mRNA is administered to a mammal together with a guide RNA that directs the mammal's TTR gene into In a pharmaceutical composition comprising lipid nanoparticles, an indel is formed at the TTR locus in at least 70% or at least 90% of the genomic DNA obtained from mammalian hepatocytes.
74. Messenger ribonucleic acid (mRNA) according to any one of claims 64 to 73, characterized in that when the mRNA is administered to a mammal together with a guide RNA that directs the mammal's TTR gene in a pharmaceutical composition comprising lipid nanoparticles, the concentration of TTR in the mammalian serum is reduced by at least 70% or at least 90%.
75. Messenger ribonucleic acid (mRNA), according to claim 62, 63, 71 or 72, characterized in that the animal is a mouse and the guide RNA has a sequence consisting of SEQ ID NO: 42.
76. Messenger ribonucleic acid (mRNA), according to claim 62, 63, 71 or 72, characterized in that the animal is a mouse and the guide RNA has a sequence consisting of SEQ ID NO: 69.
77. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the mRNA comprises a sequence with at least 90% identity with any of SEQ ID NOs: 43, 44, 51, 53 , 55-61 or 176-185.
78. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the mRNA comprises a sequence with at least 95% identity with any of SEQ ID NOs: 43, 44, 51, 53 , 55-61 or 176-185.
79. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the mRNA comprises a sequence with at least 98% identity with any of SEQ ID NOs: 43, 44, 51, 53 , 55-61 or 176-185.
80. Messenger ribonucleic acid (mRNA) according to any one of the preceding claims, characterized in that the mRNA comprises a sequence with at least 99% identity with any of SEQ ID NOs: 43, 44, 51, 53 , 55-61 or 176-185.
81. Messenger ribonucleic acid (mRNA), according to any one of the preceding claims, characterized in that the mRNA comprises a sequence with 100% identity with any of SEQ ID NOs: 43, 44, 51, 53, 55 -61 or 176-185.
82. Expression construct, characterized in that it comprises a promoter operably linked to a sequence encoding an mRNA as defined in any one of the preceding claims.
83. Plasmid, characterized in that it comprises the expression construct as defined in claim 82.
84. Host cell, characterized in that it comprises the expression construct as defined in claim 82, or the plasmid as defined in claim 83.
85. Method for preparing an mRNA, characterized in that it comprises contacting the expression construct as defined in claim 82, or the plasmid as defined in claim 83, with an RNA polymerase under permissive conditions for mRNA transcription.
86. Method according to claim 85, characterized in that the contact step is carried out in vitro.
87. Composition, characterized in that it comprises an mRNA as defined in any one of claims 1 to 81, and at least one guide RNA.
88. Lipid nanoparticle, characterized in that it comprises an mRNA as defined in any one of claims 1 to 81.
89. Pharmaceutical composition, characterized in that it comprises an mRNA as defined in any one of claims 1 to 81, and a pharmaceutically acceptable carrier.
90. Lipid nanoparticle, according to claim 88, or pharmaceutical composition, according to claim 89, characterized in that it further comprises at least one guide RNA.
91. Composition or lipid nanoparticle, according to any one of claims 87 to 90, characterized in that the at least one guide RNA directs TTR.
92. Method for editing or modifying a genome of a target gene, characterized in that it comprises contacting a cell with the mRNA, expression construct, composition or lipid nanoparticle as defined in any one of claims 1 to 83 or 87 to 91.
93. Use of mRNA, expression construct, composition or lipid nanoparticle, as defined in any one of claims 1 to 83 or 87 to 91, characterized in that it is for genome editing or modification of a target gene.
94. Use of mRNA, expression construct, composition or lipid nanoparticle, as defined in any one of claims 1 to 83 or 87 to 91, characterized in that it is for the manufacture of a drug for editing or modifying the genome of a target gene.
95. Method or use according to any one of claims 92 to 94, characterized in that the editing or modification of the target gene genome takes place in a liver cell.
96. Method or use, according to claim 95, characterized in that the liver cell is a hepatocyte.
97. Method or use, according to any one of claims 92 to 96, characterized in that the editing or modification of the target gene genome is in vivo.
98. Method or use, according to any one of claims 92 to 97, characterized in that the editing or modification of the target gene genome is in an isolated or cultured cell.

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