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    • 专利摘要:
    the present invention relates to a genome editing system for site-directed modification of a target sequence in the genome of a cell, which comprises a variant of the cas9 nuclease having greater specificity compared to the wild type cas9 nuclease or its expression construct, and an expression construct comprising the coding sequence for trna-grna fusion or ribozyme-grna fusion, or comprises an expression construct comprising the coding sequences for the nuclease variant cas9 and trna-grna or ribozyme- grna. a method is also provided to genetically modify a cell by introducing the genome editing system, which has high efficiency and high specificity.
    公开号:BR112019017138A2
    申请号:R112019017138
    申请日:2018-02-22
    公开日:2020-04-14
    发明作者:Gao Caixia；Zhang Dingbo；Zhang Huawei
    申请人:Inst Genetics & Developmental Biology Cas；
    IPC主号:
    专利说明:

    Descriptive Report of the Invention Patent for GENOME SYSTEM AND EDITING METHOD.
    FIELD OF THE INVENTION [001] The present invention relates to the field of genetic engineering. In particular, the present invention relates to a method of genome editing with high efficiency and high specificity. More specifically, the present invention relates to a method for increasing the efficiency of site-directed modification of a target sequence in an organism's genome by a highly specific Cas9 nuclease variant.
    BACKGROUND OF THE INVENTION [002] Short palindromic repetitions grouped and regularly interspersed and the CRISPR associated system (CRISPR / Cas9) is the most popular tool for genome editing. In the system, Cas9 protein cleaves a specific DNA sequence under the guidance of a gRNA to create a double strand break (DSB). DSB can activate mechanisms of intracellular repair of non-homologous tip splicing (NHEJ) and homologous recombination (HR) to repair DNA damage in cells so that the specific DNA sequence is edited during the repair process. Currently, the most commonly used Cas9 protein is Cas9 derived from Streptococcus pyogenes (SpCas9). A disadvantage of the CRISPR / Cas9 genome editing system is its low specificity and off-target effect, which greatly limits its application.
    [003] There remains a need in the technique for a method and tool that allows efficient and highly specific genome editing.
    SUMMARY OF THE INVENTION [004] In one aspect, the present invention provides a genome editing system for site-directed modification of a se
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    2/30 target sequence in the genome of a cell, comprising at least one selected from i) to iii) which follow:
    a variant of Cas9 nuclease and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion;
    an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion; and an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a tRNA-guide RNA fusion;
    where the Cas9 nuclease variant has greater specificity compared to the wild type Cas9 nuclease, where the 5 'end of the guide RNA is linked to the 3' end of the tRNA, where the fusion is cleaved at the 5 'end of the RNA -guide after being transcribed into the cell, thereby forming a guide RNA that does not carry extra nucleotide at the 5 'end.
    [005] In a second aspect, the present invention provides a genome editing system for site-directed modification of a target sequence in the genome of a cell, which comprises at least one selected from i) to iii) which follow:
    a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion;
    an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion; and
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    3/30 an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a ribozyme-guide RNA fusion;
    where the Cas9 nuclease variant has greater specificity compared to the wild type Cas9 nuclease, where the 5 'end of the guide RNA is linked to the 3' end of a first ribozyme, where the first ribozyme is designed to cleave the fusion at the 5 'end of the guide RNA, thereby forming an RNA guide that does not carry extra nucleotide at the 5' end.
    [006] In a third aspect, the present invention provides a method for genetically modifying a cell, which comprises introducing the genome editing system of the present invention into the cell, so that the Cas9 nuclease variant is targeted to a target in the cell's genome by the guide RNA, and results in replacement, deletion and / or addition of one or more nucleotides in the target sequence.
    [007] In a fourth aspect, the present invention provides a genetically modified organism, which comprises a genetically modified cell produced using the method of the present invention.
    BRIEF DESCRIPTION OF THE DRAWINGS [008] Figure 1 shows strategies for sgRNA design for target sequences with different 5 'end nucleotides when using U3 or U6. A: through fRNA fusion, sgRNA can be designed without considering the 5 'end nucleotide of the target sequence; B: precise divination of tRNA-sgRNA fusion.
    [009] Figure 2 shows the editing efficiency of WT SpCas9 (SpCas9 wild type), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9HF1 on class targets (1).
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    4/30 [0010] Figure 3 shows the editing efficiency of WT SpCas9 (wild type SpCas9), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9HF1 on class targets (2).
    [0011] Figure 4 shows that the additional nucleotide at the 5 'end of sgRNA affects the editing efficiency when U6 promoter is used.
    [0012] Figure 5 shows that for the OsMKK4 locus, tRNA-sgRNA can improve editing efficiency and maintain high specificity compared to sgRNA.
    [0013] Figure 6 shows that for the OsCDKB2 locus, the use of tRNA-sgRNA can increase the editing efficiency for the level of wild type SpCas9, while maintaining high specificity.
    [0014] Figure 7 shows the editing specificity of the Cas9 variant for incompatibility between gRNA and target sequence.
    [0015] Figure 8 shows that tRNA-sgRNA improved the editing efficiency of eSPCas9 (1.1) and SpCas9-HF1 to that of wild-type SpCas9 in human cells.
    [0016] Figure 9 shows the pUC57-U3-tRNA-sgRNA vector sequence structure for tRNA-sgRNA fusion expression.
    DETAILED DESCRIPTION OF THE INVENTION
    1. Definition [0017] In the present invention, unless otherwise indicated, the scientific and technological terminologies used herein refer to meanings commonly understood by one skilled in the art. Also, the terminologies and experimental procedures used here with respect to protein and nucleotide chemistry, molecular biology, tissue cell culture, microbiology, immunology, all belong to conventional terminologies and methods commonly used in the art. For example, the recombination of standard DNA and molecular cloning technology used here is well known
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    5/30 are used in the art and are described in detail in the references that follow: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory ManualCold Spring Harbor Laboratory PressCold Spring Harbor, 1989. In the meantime, in order To better understand the present invention, definitions and explanations for the relevant terminologies are provided below.
    [0018] Nuclease Cas9 and Cas 9 can be used interchangeably here, which refer to an RNA-directed nuclease, including the Cas9 protein or fragments thereof (such as a protein comprising a Cas9 active DNA dividing domain and / or a Cas9 gRNA binding domain). Cas9 is a component of the CRISPR / Cas genome editing system (short palindromic repeats grouped and regularly interspersed and its associated system), which targets and cleaves a target DNA sequence to form double stranded DNA (DSB) breaks under the guidance of a guide RNA.
    [0019] Guide RNA and gRNA can be used interchangeably here, which are typically composed of crRNA and tracrRNA molecules forming complexes through partial complement, where crRNA comprises a sequence that is sufficiently complementary to a target sequence for hybridization and directs the CRISPR complex (Cas9 + crRNA + tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single RNA guide (sgRNA) can be designed, which comprises the characteristics of both crRNA and tracrRNA.
    [0020] As used here, the terms tRNA and transfer RNA are used interchangeably to refer to small molecule RNAs that have the function of loading and transporting amino acids. The tRNA molecule usually consists of a short chain of about 70-90 nucleotides folded into a tre
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    6/30 vo. In eukaryotes, tRNA genes in the genome are transcribed into tRNA precursors, which are then processed into mature tRNA after excision of the additional 5 'and 3' sequences by RNase P and RNase Z.
    [0021] As used here, the term ribozyme refers to an RNA molecule that has a catalytic function that participates in the dividing and processing of RNA through catalysis of the hydrolysis reactions of the phosphodiester and phosphodiester link.
    [0022] Genome as used here comprises not only chromosomal DNA present in the nucleus, but also organelle DNA present in the subcellular components (eg, mitochondria, plastids) of the cell.
    [0023] As used here, an organism includes any organism that is suitable for genomic editing. Exemplary organisms include, but are not limited to, mammals such as human, mouse, rat, monkey, dog, pig, sheep, cattle, cat; birds such as chicken, duck, goose; plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis and the like.
    [0024] Genetically modified organism or genetically modified cell means an organism or cell that contains an exogenous polynucleotide or modified gene or expression control sequence within its genome. For example, the exogenous polynucleotide is stably integrated into the genome of an organism or cell and inherited by successive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. The modified expression control gene or sequence is the sequence in the genome of the organism or cell that comprises single or multiple deoxynucleotide substitutions, deletions and additions.
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    7/30 [0025] The term exogenous with respect to the sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.
    [0026] Polynucleotide, nucleic acid sequence, nucleotide sequence and nucleic acid fragment are used interchangeably to refer to a single or double-stranded RNA or DNA polymer, optionally containing synthetic, unnatural or altered nucleotide bases . Nucleotides (usually found in their 5'-monophosphate form) are referred to by their unique letter designation as follows: A for adenylate or deoxyadenylate (for RNA or DNA, respectively), C for cytidylate or deoxycytidate, G for guanylate or deoxyguanilate, U for uridylate, T for deoxythymidylate, R for purines (A or G), Y for pyrimidines (C or T), K for G or T, Ή for A or C or T, I for inosine and N for any nucleotide.
    [0027] Polypeptide, peptide, amino acid sequence and protein are used here interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers where one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The terms polypeptide, peptide, amino acid sequence and protein are also inclusive of modifications including, but not limited to, glycosylation, lipid bonding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
    [0028] As used herein, an expression construct refers to a vector suitable for expression of a nucleotide sequence of interest in an organism, such as a recombinant vector. Ex
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    8/30 pressure refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (such as transcribed to produce a functional mRNA or RNA) and / or translation of RNA into a protein precursor or a mature protein.
    [0029] Expression construct of the invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector or, in some embodiments, an RNA that can be translated (such as an mRNA).
    [0030] Expression construct of the invention may comprise regulatory sequences and nucleotide sequences of interest that are derived from different sources, or regulatory sequences and nucleotide sequences of interest derived from the same source, but arranged in a different way than normally found in nature .
    [0031] Regulatory sequence and regulatory element are used interchangeably and refer to nucleotide sequences located upstream (non-coding sequence 5 '), inside, or downstream (non-coding sequences 3') of a coding sequence, and which influence the transcription, processing or stability of RNA or translation of the associated coding sequence. Regulatory sequences can include, but are not limited to, promoters, leader translation sequences, introns and polyadenylation recognition sequences.
    [0032] Promoter refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, the promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. The promoter can be a constitutive promoter or a tissue specific promoter or a promoter of
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    9/30 senvolvementally regulated or an inducible promoter.
    [0033] Constitutive promoter refers to a promoter that can cause expression of a gene in most circumstances in most cell types. Tissue specific promoter and preferred tissue promoter are used interchangeably and refer to a promoter that is expressed predominantly, but not necessarily exclusively, in a tissue or organ, but that can also be expressed in a specific cell or cell type. Developmentally regulated promoter refers to a promoter whose activity is determined by developmental events. Inducible promoter selectively expresses a DNA sequence operably linked to it in response to an endogenous or exogenous stimulus (environmental, hormones or chemical signals, and others).
    [0034] As used herein, the term operably linked means that a regulatory element (for example, but not limited to, a promoter sequence, a transcription termination sequence, and the like) is associated with a nucleic acid sequence (such as a coding sequence or an open reading frame), so that the transcription of the nucleotide sequence is controlled and regulated by the regulatory transcriptional element. Techniques for operably linking a region and regulatory element to a nucleic acid molecule are known in the field.
    [0035] Introduction of a nucleic acid molecule (eg, plasmid, linear nucleic acid fragment, RNA, etc.) or protein into an organism means that the nucleic acid or protein is used to transform a cell in the organism so that the nucleic acid or protein works in the cell. As used in the present invention, transformation includes both stable and transient transformations. Stable transformation refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in inheritance
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    Stable 10/30 of foreign genes. Once stably transformed, the exogenous nucleic acid sequence is adequately integrated into the organism's genome and any of its successive generations. Transient transformation refers to the introduction of a nucleic acid or protein molecule into a cell, carrying out its function without the stable inheritance of an exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.
    2. Genome editing system with high efficiency and high specificity [0036] The nuclease variant Cas9 eSpCas9 (1.0) (K810A / K1003A / R1060A), eSpCas9 (1.1) (K848A / K1003A / R1060A) by Feng Zhang and others and the Cas9 nuclease variant SpCas9-HF1 (N497A / R661A / Q695A / Q926A) developed by J. Keith Joung and others are able to significantly reduce the off-target rate in genomic editing, and therefore have high specificity. However, surprisingly, the present inventors have found that these three variants of Cas9 nuclease, while having high specificity, have a much lower gene editing efficiency compared to wild type Cas9.
    [0037] The present inventors have surprisingly found that by fusing the 5 'end of the guide RNA to a tRNA, the editing efficiency of the high specificity Cas9 nuclease variant can be increased, even at the wild type level, while maintaining the high specificity.
    [0038] Without wishing to be limited by any theory, it is believed that the reduction in the editing efficiency of high specificity Cas9 nuclease variants is related to whether guide RNA transcription can be precisely initiated or not. In the art, promoters commonly used for in vivo guide RNA production include,
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    11/30 for example, U6 or U3 snRNA promoters, for which transcription is directed by RNA III polymerase. The U6 promoter needs to initiate transcription in G, and then for target sequences with the first nucleotide of A, C or T, an additional G will be present at the 5 'end of sgRNA as the transcript. The U3 promoter initiates transcription in A, and then for target sequences with the first nucleotide of G, C or T, an additional A will be present at the 5 'end of sgRNA as the transcript. The inventors have found that the editing efficiency of high specificity Cas9 nuclease variants is reduced if an additional nucleotide is present at the 5 'end of the sgRNA. Through fusion transcription with a tRNA, due to the precise tRNA processing mechanism (precise removal of additional 5 'and 3' sequence of tRNA precursor to form mature tRNA), sgRNA without additional nucleotide at the 5 'end can be readily obtained even using U6 or U3 promoters, without the need to consider the type of the first nucleotide of the target sequence. In this way, the editing efficiency of high specificity Cas9 nuclease variants can be improved, and the selectable range of target sequences can be extended. Still, without intending to be limited by theory, fRNA with tRNA can increase the level of sgRNA expression, which can also contribute to improving the editing efficiency of high specificity Cas9 nuclease variants.
    [0039] In this way, the present invention provides a genome editing system for site-directed modification of a target sequence in the genome of a cell, which comprises at least one selected from i) to iii) which follow:
    a variant of Cas9 nuclease and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion;
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    12/30 an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion; and an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a tRNA-guide RNA fusion;
    where the Cas9 nuclease variant has greater specificity compared to the wild type Cas9 nuclease, where the 5 'end of the guide RNA is linked to the 3' end of the tRNA, where the fusion is cleaved at the 5 'end of the RNA -guide after being transcribed into the cell, thereby forming a guide RNA that does not carry extra nucleotide at the 5 'end.
    [0040] In some modalities, the tRNA and the cell to be modified are of the same species.
    [0041] In some specific embodiments, the tRNA is encoded in the following sequence: aacaaagcaccagtggtctagtggtagaatagtaccc tgccacggtacagacccgggttcgattcccggctggtgca (SEQ ID NO: 1).
    [0042] The design of the fusion of tRNA-guide RNA is within the skill of the person skilled in the art. For example, reference can be made to Xie et al., PNAS, Mar 17, 2015; vol. 112, no. 11.3570-3575.
    [0043] The present invention also considers the fusion of a guide RNA and a ribozyme. Based on what is found in the invention that the editing efficiency of high specificity Cas9 nuclease variants is related to the initiation of accurate sgRNA transcription, by using the ribozyme ability to cut RNA at a specific site, it is possible to produce sgRNA without additional nucleotide at the 5 'end through rational design of a fusion of RNA and ribozyme, in order to improve editing efficiency while maintaining the
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    13/30 high specificity.
    [0044] In this way, the invention also provides a genome editing system for site-directed modification of a target sequence in the genome of a cell, which comprises at least one selected from i) to iiii) which follow:
    a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion;
    an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion; and an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a ribozyme-guide RNA fusion;
    where the Cas9 nuclease variant has greater specificity compared to the wild type Cas9 nuclease, where the 5 'end of the guide RNA is linked to the 3' end of a first ribozyme, where the first ribozyme is designed to cleave the fusion at the 5 'end of the guide RNA, thereby forming an RNA guide that does not carry extra nucleotide at the 5' end.
    [0045] In one embodiment, the 3 'end of the guide RNA is linked to the 5' end of a second ribozyme, the second ribozyme is designed to cleave the fusion at the 3 'end of the guide RNA, thereby forming an RNA- guide that does not carry extra nucleotide at the 3 'end.
    [0046] The design of the first ribozyme or the second ribozyme is within the skill of the person skilled in the art. For example, reference can be made to Gao and others, JIPB, Apr, 2014; Vol 56, Edition 4, 343 Petition 870190079766, of 16/08/2019, p. 27/110
    14/30
    349.
    [0047] In a specific embodiment, the first ribozyme is encoded by the following sequence: 5 '- (N) 6CTGATGAGTCCGTGAGGA CGAAACGAGTAAGCTCGTC-3' (SEQ ID NO: 12), where N is independently selected from A, G, C and T and (Ν) θ refers to a sequence reversely complementary to the first 6 nucleotides at the 5 'end of the guide RNA. In a specific embodiment, the second ribozyme is encoded by the following sequence: 5'-GGCCG GCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCT TCGGCATGGCGAATGGGAC-3 ’(SEQ ID NO: 13).
    [0048] The Cas9 nuclease variant in the invention which has greater specificity compared to wild type Cas9 nuclease can be derived from Cas9 of several species, for example, Cas9 derived from Streptococcus pyogenes (SpCas9, nucleotide sequence shown in SEQ ID NO : 2, amino acid sequence shown in SEQ ID NO: 3).
    [0049] In some embodiments of the invention, the nuclease variant Cas9 is a variant of SEQ ID NO: 2, which comprises an amino acid substitution at position 855 of SEQ ID NO: 2. In some specific embodiments, the amino acid substitution in position 855 is K855A.
    [0050] In some embodiments of the invention, the nuclease variant Cas9 is a variant of SEQ ID NO: 2, which comprises amino acid substitutions at positions 810, 1003 and 1060 of SEQ ID NO: 2. In some specific embodiments, the substitutions amino acids are K810A, K1003A and R1060A respectively.
    [0051] In some embodiments of the invention, the nuclease variant Cas9 is a variant of SEQ ID NO: 2, which comprises amino acid substitutions at positions 848, 1003 and 1060 of SEQ ID NO: 2. In some specific embodiments, the replacements
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    15/30 amino acids are K848A, K1003A and R1060A respectively.
    [0052] In some embodiments of the invention, the nuclease variant Cas9 is a variant of SEQ ID NO: 2, which comprises amino acid substitutions at positions 611, 695 and 926 of SEQ ID NO: 2. In some specific embodiments, the substitutions amino acids are R611 A, Q695A and Q926A respectively.
    [0053] In some embodiments of the invention, the nuclease variant Cas9 is a variant of SEQ ID NO: 2, which comprises amino acid substitutions at positions 497, 611, 695 and 926 of SEQ ID NO: 2. In some specific embodiments, amino acid substitutions are N497A, R611 A, Q695A and Q926A respectively.
    [0054] In some specific embodiments of the invention, the nuclease variant Cas9 comprises an amino acid sequence as shown in SEQ ID NO: 4 (eSpCas9 (1.0)), SEQ ID NO: 5 (eSpCas9 (1.1)) or SEQ ID NO : 6 (SpCas9-HF1).
    [0055] In some embodiments of the invention, the Cas9 nuclease variant of the invention further comprises a nuclear localization sequence (NLS). In general, one or more NLSs in the Cas9 nuclease variant must have sufficient strength to drive the accumulation of the Cas9 nuclease variant in the cell nucleus in an amount sufficient for the genome editing function. In general, the resistance of nuclear localization activity is determined by the number and position of NLSs, and one or more specific NLSs used in the Cas9 nuclease variant, or a combination thereof.
    [0056] In some embodiments of the present invention, the NLSs of the Cas9 nuclease variant of the invention may be located at the N-terminus and / or the C-terminus. In some embodiments, the Cas9 nuclease variant comprises about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the Cas9 nuclease variant comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or
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    16/30 near the N terminal. In some embodiments, the Cas9 nuclease variant comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C terminal. In some embodiments, the Cas9 nuclease variant comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. Where there is more than one NLS, each NLS can be selected as independent from other NLSs. In some preferred embodiments of the invention, the Cas9 nuclease variant comprises two NLSs, for example, the two NLSs are located at the N-terminus and the C-terminus, respectively.
    [0057] In general, NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of a protein, but other types of NLS are also known in the art. Non-limiting examples of NLSs include KKRKV (nucleotide sequence 5'-AAGAAGAGAAAGGTC-3 '), PKKKRKV (nucleotide sequence 5'-CCCAAGAAGAAGAGGAAGGTG-3' or CCAAAGA AGAAGAGGAAGGTT) or SGGSPGKGAGGAGG .
    [0058] In some embodiments of the invention, the N-terminus of the nuclease variant Cas9 comprises an NLS with an amino acid sequence shown by PKKKRKV. In some embodiments of the invention, the C-terminus of the Cas9 nuclease variant comprises an NLS with an amino acid sequence shown by SGGSPKKKRKV.
    [0059] Furthermore, the Cas9 nuclease variant of the present invention can also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, and the like, depending on the location of the DNA to be edited .
    [0060] To obtain effective expression in the target cell, in addition
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    In some embodiments of the invention, the nucleotide sequence encoding the Cas9 nuclease variant is optimized at the codon for the organism from which the cell to be edited in the genome originates.
    [0061] Codon optimization refers to a process of modifying a nucleic acid sequence for increased expression in host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with codons that are most often or most often used in the genes of that host cell while maintaining the native amino acid sequence. Several species exhibit a particular tendency for certain codons of a particular amino acid. Codon tendency (differences in codon usage between organisms) is often related to the translation efficiency of messenger RNA (mRNA), which is in turn believed to depend, among other things, on the properties of the codons being translated and the availability of molecules transfer RNA (tRNA). The predominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. In this way, genes can be made especially for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / and these tables can be adapted in several ways. See Nakamura, Y. and others, Codon usage tabulated from the international DNA sequence databases: status for the year 2000 Nucl. Acids Res. 28: 292 (2000).
    [0062] The organism, from which the cell that can be edited in the genome by the system of the invention is derived, includes, but is not limited to, mammals such as human, mice, rat, monkey,
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    18/30 dog, pig, sheep, cow and cat; birds such as chicken, duck and goose; plants including monocots and dicots, for example, rice, corn, wheat, sorghum, barley, soybeans, peanuts and Arabidopsis thaliana and the like.
    [0063] In some specific embodiments of the invention, the optimized nucleotide sequence in the codon encoding the Cas9 nuclease variant is as shown in SEQ ID NO: 7 (eSpCas9 (1.0)), SEQ ID NO: 8 (eSpCas9 (1.1)) or SEQ ID NO: 9 (SpCas9-HF1).
    [0064] In some embodiments of the invention, the guide RNA is a single guide RNA (sgRNA). Methods of constructing suitable sgRNAs according to a given target sequence are known in the art. See, for example, Wang, Y. and others, Simultaneous editing of three homoeoalleles in hexapioid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947-951 (2014); Shan, Q. et al., Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686-688 (2013); Liang, Z. et al., Targeted mutagenesis in Zea mays using TALENs and the CRISPR / Cas system. J Genet Genomics. 41,63-68 (2014).
    [0065] In some embodiments of the invention, the nucleotide sequence encoding the Cas9 nuclease variant and / or the nucleotide sequence encoding the guide RNA fusion are operably linked to an expression regulatory element such as a promoter.
    [0066] Examples of promoters that can be used in the present invention include, but are not limited to, polymerase (pol) I, pol II or pol III promoters. Examples of pol I promoters include chicken RNA pol I promoter. Examples of pol II promoters include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, sarcoma rous virus (RSV-LTR) long terminal repeat promoter and simian virus 40 (SV40) immediate early promoter. Examples of pol III promoters include U6 promoter and
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    19/30 H1 promoter. Inducible promoter such as metallothionein promoter can be used. Other examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, βgalactosidase promoter and bacteriophage Sp6 promoter, etc. When used for plants, promoters that can be used include, but are not limited to, cauliflower mosaic virus promoter 35S, corn promoter Ubi-1, wheat promoter U6, rice promoter U3, corn promoter U3 and promoter of rice actin, etc.
    3. Method for genetically modifying a cell [0067] In another aspect, the invention provides a method for genetically modifying a cell, which comprises: introducing the genome editing system of the invention into the cell, thus the nuclease variant Cas9 it is directed to the target sequence in the cell's genome by the guide RNA, and results in the replacement, deletion and / or addition of one or more nucleotides in the target sequence.
    [0068] The design of the target sequence that can be recognized and directed by a complex of cas9 and guide RNA is within the technical skills of a common expert in the technique. In general, the target sequence is a sequence that is complementary to a leader sequence of about 20 nucleotides comprised in guide RNA, and whose 3 'end is immediately adjacent to the adjacent protospacer (PAM) NGG motif.
    [0069] For example, in some embodiments of the invention, the target sequence has the structure: 5'-Nx-NGG-3 ', where N is selected independently from A, G, C and T; X is an integer of 14 <X <30; NX represents X contiguous nucleotides and NGG is a PAM sequence. In some specific embodiments of the invention, X is 20.
    [0070] In the present invention, the target sequence to be modified can be located anywhere in the genome, for example, within a functional gene such as a gene encoding pro
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    The protein or, for example, can be located in a regulatory region of gene expression such as a promoter region or an enhancer region, and thus carry out the functional modification of said gene or carry out the modification of expression of a gene.
    [0071] Substitution, deletion and / or addition of the target cell sequence can be detected by T7EI, PCR / RE or sequencing methods, see, for example, Shan, Q., Wang, Y, Li, J. & Gao, C. Genome editing in rice and wheat using the CRISPR / Cas system. Nat. Protoc. 9, 2395-2410 (2014).
    [0072] In the method of the present invention, the genome editing system can be introduced into the cell using various methods well known to the person skilled in the art.
    [0073] Methods for introducing the genome editing system of the present invention into the cell include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as a baculovirus , a vaccinia virus, an adenovirus or other viruses), particle bombardment, PEG-mediated protoplast transformation or agrobacterium-mediated transformation.
    The cell that can be subjected to genome editing with the method of the present invention can be, for example, mammals such as human, mouse, rat, monkey, dog, pig, sheep, cow, cat; birds such as chicken, duck and goose; plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis thaliana, etc.
    [0075] In some embodiments, the method of the present invention is carried out in vitro. For example, the cell is an isolated cell. In some other embodiments, the method of the present invention can be performed in vivo. For example, the cell is a cell within an organism, and the system of the present invention can be introduced in vi
    Petition 870190079766, of 16/08/2019, p. 34/110
    21/30 vo in said cell using, for example, a virus-mediated method. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a somatic cell.
    [0076] In another aspect, the present invention further provides a genetically modified organism comprising a genetically modified cell produced using the method of the present invention.
    [0077] The organism includes, but is not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cows, cats; birds such as chickens, ducks and geese; plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts and Arabidopsis thaliana.
    Examples
    Materials and methods
    Construction of Binary Expression Vectors pJIT163-SpCas9, pJIT163-eSpCas9 (1.0), pJIT163-eSpCas9 (1.1) and pJIT163-SpCas9-HF1 [0078] Sequences of SpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 optimized at the codon for rice. SpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 were obtained through site-directed mutagenesis using Fast MultiSite Mutagenesis System (TransGen) with plasmid pJIT163-SpCas9 (SEQ ID NO: 10) as the model.
    Construction of sgRNA Expression Vector [0079] Target sequences of sgRNA used in the experiments are shown in Table 1 as follows:
    Table 1. Target gene and sgRNA target sequence
    sgRNA Target string Oligo-F Oligo-R OSCDKB2 AGGTCGGGGA GGGGACGTAC GGG GGCAAGGTCGGGGAGG GGACGTAC AAACGTACGTCCCCTCCCCG ACCT OsMKK4 GACGTCGGCGAGGAAGGCCT CGG GGCAGACGTCGGCGAGG AAGGCCT AAACAGGCCTTCCTCGCCGA CGTC TO 1 CATGGTGGGGAAAGCTTGGA GGCACATGGTGGGGAAA GCTTGGA AAACTCCAAGCTTTCCCCAC CATG
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    22/30
    GGG A2 CCGGACGACGACGTCGAC-GACGG GGCACCGGACGACGACG TCGACGA AAACTCGTCGACGTCGTCGT CCGG A3 TTGAAGTCCCTTCTAGATGGAG G GGCATTGAAGTCCCTTCT AGATGG AAACCCATCTAGAAGGGACT TCAA A4 ACTGCGACAC CCAGATATCG TGG GGCAACTGCGACACCCA GATATCG AAACCGATATCTGGGTGTCGC AGT PDS GTTGGTCTTTG CTCCTGCAGA GG GGCAGTTGGTCTTTGCTC CTGCAG AAACCTGCAGGAGCAAAGAC CAAC
    [0080] sgRNA expression vectors: pOsU3-CDKB2-sgRNA, pOsU3-MKK4-sgRNA, pOsU3-A1-sgRNA as well as pOsU3-A2sgRNA, pOsU3-A3-sgRNA, pOsU3-A4-sgRNA and pOsU3-PDG constructed based on pOsU3-sgRNA (Addgene ID53063) as previously described (Shan, Q. et al., Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31,686-688, 2013).
    Construction of tRNA-sqRNA Expression Vectors [0081] tRNA-sgRNA expression vectors are constructed based on the vectorpUC57-U3-tRNA-sgRNA (SEQ ID NO: 11, Figure 6). A linear vector is obtained after digestion of pUC57-U3-tRNA-sgRNA with Bsal, the corresponding oligo-F and oligo-R are annealed and connected to the linear vector, and the subsequent steps are similar for the construction of the sgRNA expression vectors .
    Table 2. Target Genes and Nucleotide Sequences for Construction of tRNA-sgRNA Expression Vectors
    sgRNA Target string Oligo-F Oligo-R OsCDKB2 AGGTCGGGGAGGGGACGTACGGG TGCAAGGTCGGGGAGGGGACGTAC AAACGTACGTCCCCTCCCCGACCT OsMKK4 GACGTCGGCGAGGAAGGCCTCGG TGCAGACGTCGGCGAGGAAGGCCT AAACAGGCCTTCCTCGCCGACGTC TO 1 CATGGTGGGGAAAGCTTGGAGGG TGCACATGGTGGGGAAAGCTTGGA AAACTCCAAGCTTTCCCCACCATG A2 CCGGACGACGACGTCGACGACGG TGCACCGGACGACGACGTCGACGA AAACTCGTCGACGTCGTCGTCCGG A3 TTGAAGTCCCTTCT TGCATTGAAGTCCCTT AAACCCATCTAGAAGGGACT
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    23/30
    AGATGGAGG CTAGATGG TCAA A4 ACTGCGACACCCAGATATCGTGG TGCACTGCGACACCCAGATATCG AAACCGATATCTGGGTGTCGCAGT PDS GTTGGTCTTTGCTCCTGCAGAGG TGCAGTTGGTCTTTGCTCCTGCAG AAACCTGCAGGAGCAAAGACCAAC
    Protoplast tests [0082] Nipponbare rice cultivar is used in the research. Protoplast transformation is carried out as described below. Transformation is performed with 10 pg of each plasmid via PEG-mediated transfection. Protoplasts were collected after 48 h and DNA was extracted for PCR-RE assay.
    Preparation and processing of rice protoplasts
    1) Leaf sheath of the seedlings was used for protoplast isolation and cut to about 0.5 mm wide with a sharp blade;
    2) Immediately after the incision, transferred to 0.6M Mannitol solution and left in the dark for 10 min;
    3) Mannitol solution was removed by filtration and the products were transferred to an enzyme solution and evacuated for 30 min;
    4) Enzymolysis was carried out for 5-6 h in the dark with gentle agitation (decolorization shaker, speed 10);
    5) After the end of the enzymolysis, an equal volume of W5 was added, horizontal shaking for 10 s to release protoplasts;
    6) Protoplasts were filtered in a 50 ml round bottom centrifuge tube with a 40 pm nylon membrane and washed with W5 solution;
    7) Horizontal centrifugation 250 g for 3 min to precipitate protoplasts, the supernatant was discarded;
    8) Protoplasts were resuspended by adding 10 ml of W5 and then centrifuged at 250 g for 3 min and the supernatant was
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    24/30 card;
    9) An appropriate amount of MMG solution was added to resuspend the protoplasts to a concentration of 2x10 6 / ml;
    Note: all the steps above were performed at room temperature;
    10) 10-20 pg of plasmid, 200 pl of protoplasts (about 4x10 5 cells) and 220 μΙ of fresh PEG solution were added to a 2 ml centrifuge tube, mixed and placed at room temperature in the dark for 10- 20 minutes to induce transformation;
    11) After finishing the transformation, 880 μΙ of W5 solution was slowly added, and the tubes were gently turned upside down for mixing, centrifuged horizontally 250 g for 3 min and the supernatant was discarded;
    12) The products were resuspended in 2 ml of Wl solution, transferred to a six-well plate, grown at room temperature (or 25 ° C) in the dark. For extraction of genomic DNA from protoplasts, the products had to be grown for 48 h. Mutation Identification by Deep Sequencing
    Deep sequencing analysis is performed with reference to Liang, Z., Chen, K., Li, T, Zhang, Y., Wang, Y., Zhao, Q., Liu, J., Zhang, H., Liu, C., Ran, Y., and others, (2017). Efficient DNA-free genome editing of bread wheat using CRISPR / Cas9 ribonucleoprotein complexes. Nature Communications 8, 14261.
    Example 1: Comparison of editing capabilities of WT SpCas9 and variants of it for target sites [0083] WT SpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 were respectively constructed in a transient expression vector pJIT163, and the expressions of WT SpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 are driven by a promoter of the gene
    Petition 870190079766, of 16/08/2019, p. 38/110
    25/30 of the corn ubiquitin. sgRNAs were constructed in the pOsU3sgRNA vector and the expression of sgRNAs is driven by the OsU3 promoter. Rice protoplasts were transformed, and protoplast DNA was extracted for PCR-RE analysis to assess the mutation efficiency. Five target sites (A1, A2, A3, A4 and PDS, see Figure 2 and Figure 3) are selected to compare the difference in editing capabilities of wild-type SpCas9 and eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9HF1.
    [0084] The OsU3 promoter has to start transcription with nucleotide A and, thus, the design of the sgRNA expression vectors for the target sites can be divided into two conditions as follows:
    [0085] if the first nucleotide at the 5 'end of the desired sgRNA target sequence (20 bp) is either G / T / C, as the U3 promoter initiates transcription with an A, an additional A will be added to the 5' end of the transcribed sgRNA and, still, the transcribed sgRNA cannot be fully compatible with the target sequence. SgRNA expression vector can be constructed as U3 + AN20 in Figure 1, while N20 is the target sequence, A is the additional nucleotide at the 5 'end;
    [0086] If the first nucleotide at the 5 'end of the desired sgRNA target sequence (20 bp) is A, it can be used by the U3 promoter for initiation of transcription, and thus no additional nucleotides will exist at the 5' end of the transcribed sgRNA. SgRNA expression vector can be constructed as U3 + AN19 in Figure 1, while AN19 is the target sequence.
    [0087] The selected target sites A1, A2, A3 and PDS belong to the target sites of class (1), and the target site A4 belongs to target sites of class (2).
    [0088] The results of the experiment show (Figure 2) that the
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    26/30 eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 editing efficiencies for class (1) target sites are extremely low. The difference between the editing efficiencies of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9HF1 and the editing efficiency of WT SpCas9 is not significant for class target sites (2). This shows that the additional nucleotide at the 5 'end of the sgRNA resulting from the transcription can reduce the editing efficiencies of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1.
    [0089] Similar to the OsU3 promoter, corn U6 promoter (TaU6) has to start transcription with the G nucleotide and, thus, the design of the sgRNA expression vectors for the target sites can be divided into two conditions as follows :
    [0090] if the first nucleotide at the 5 'end of the desired sgRNA (20bp) target sequence is any of A / T / C, as the U6 promoter initiates transcription with a G, an additional G will be added to the 5' end of the sgRNA transcribed and, still, the sgRNA transcribed cannot be completely compatible with the target sequence;
    [0091] if the first nucleotide at the 5 'end of the desired sgRNA (20 bp) target sequence is G, it can be used by the U6 promoter for initiation of transcription and, therefore, no additional nucleotides will exist at the 5' end of the transcribed sgRNA .
    [0092] The OsPDS target site belongs to class target sites (2). TaU6 promoter was added to conduct the transcription of GNw and GN20, sgRNAs against OsPDS target site, where GN20 can mimic class (1) target sites, namely, with an additional G at the 5 'end of the sgRNA.
    Table 3. Target gene and oligonucleotide sequences for construction of TaU6-sgRNA expression vectors
    sgRNA Target string Oligo-F Oligo-R OsPDS-GNig GTTGGTCTTTGCTC CTGCAGAGG GGCGTTGGTCTT TGCTCCTGCAG AAACCTGCAGGAGCAAAGA CCAA OSPDS-GN20 GTTGGTCTTTGCTC CTGCAGAGG GGCGGTTGGTCTTTGCTCCTGCAG AAACCTGCAGGAGCAAAGAC CAAC
    [0093] The results show (Figure 2) that an additional G in the
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    27/30 5 'end of sgRNA significantly reduces the editing efficiency of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1.
    Example 2: Increased efficiency of adding Cas9 variants by tRNA-sgRNA fusion [0094] According to the result of Example 1, an important factor influencing the editing efficiencies of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 is whether sgRNA is precisely initiated or not.
    [0095] According to previous report, fusion of tRNA to the 5 'end of a sgRNA can over-suppress the expression of the sgRNA and result in accurate dividing at the 5' end of the sgRNA, thereby avoiding additional nucleotide at the 5 'end of the sgRNA. (See Xie, K., Minkenberg, B., Yang, Y Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA. 2015 Mar 17; 112 (11): 3570-5. Doi : 10.1073 / pnas. 1420294112. Epub 2015 Mar 2.) [0096] sgRNA for each target site in Example 1 was fused to tRNA and expressed under the control of the OsU3 promoter. Experiments were carried out using the method of Example 1 with tRNA-sgRNA instead of sgRNAs. As shown in Figure 2, for target sites A1, A2, A3 and PDS, the editing efficiencies of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 are significantly increased using tRNA-sgRNAs instead of sgRNAs.
    Example 3: influences of tRNA-sgRNA fusion for editing specificity of Cas9 variants
    3.1 Rice 0sMKK4 target site [0097] A GACGTCGGCGAGGAAGGCCTCGG target site in rice MKK4 gene was selected to design sgRNA and tRNAsgRNA. This target site has two possible off-target sites as shown in Figure 5. A vector for sgRNA or tRNA expression
    Petition 870190079766, of 16/08/2019, p. 41/110
    28/30 sgRNA and vectors for expression of WTSpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 were respectively transformed into rice protoplasts. Two days after transformation, protoplast DNA was extracted, and genomic fragments from the target site and off-target sites were amplified using specific primers. Mutation rates at the three sites were analyzed using second generation sequencing technology.
    [0098] The result of the experiment is shown in Figure 5:
    [0099] when sgRNAs were used, compared to
    WTSpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 have an extremely low off-target effect, but have significantly lower editing efficiencies.
    [00100] When tRNA-sgRNAs were used, the editing efficiency of each group was increased, however, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 can maintain relatively high specificity. Particularly for SpCas9-HF1, only extremely low level mutations can be detected for both off-target sites. In this way, the combination of tRNA-sgRNA and SpCa9-HF1 is particularly suitable for genome editing with high efficiency and high specificity.
    3.2 OsCDKB2 rice target site [00101] An AGGTCGGGGAGGGGACGTACGGG target site in the OsCDKB2 rice gene was selected to design sgRNA. This target site has three possible off-target sites as shown in Figure 6. A vector for expression of sgRNA or tRNA-sgRNA and vectors for expression of WTSpCas9, eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9HF1 were co-transformed into rice protoplasts, respectively. . Two days after transformation, protoplast DNA was extracted, and genomic fragments from the target site and off-target sites were amplified using specific primers. Mutation rates of
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    29/30 other sites were analyzed using deep sequencing.
    [00102] The results of the experiment are shown in Figure 6. The editing efficiencies of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9HF1 for the target sites are effectively increased using tRNAsgRNA instead of sgRNA. In particular, the editing efficiency of SpCas9-HF1 can be restored to a wild-type level, and high specificity can be maintained. Since this target sequence begins with an A, by which the U3 promoter can initiate transcription precisely, the increased editing efficiency may result from the increased expression level of sgRNA due to fRNA fusion.
    Example 4: Editing the specificity of Cas9 variants for incompatibility between gRNA and target sequence [00103] When designing sgRNA for a GACGTC GGCGAGGAAGGCCTCGG target in MKK4 rice gene, incompatibilities of two adjacent bases were artificially introduced (purine for purine and pyrimidine to pyrimidine). Editing under the condition that sgRNA may not be fully compatible with the target site has been detected. It is considered to be off target if editing can be detected. The experiments were carried out in a manner similar to that in Example 3.1.
    [00104] The results of the experiments were shown in Figure
    7. When tRNA-sgRNA is used, variants of SpCas9 showed greater sensitivity to incompatibilities between gRNA and the target sequence (particularly the closest incompatibility to either end).
    Example 5: Editing the efficiency and specificity of Cas9 variants in human embryonic kidney 293 cells [00105] sgRNAs were designed against a GGTGAGTGAGTGTGTGCGTGTGG target sequence within human VEGFA gene. U6: sgRNA-GNi9 and U6: tRNA-sgRNA-N2o represent that sgRNAs
    Petition 870190079766, of 16/08/2019, p. 43/110
    30/30 U6 promoter transcripts are 20 nt long and completely compatible with the target sequence; U6: sgRNA-GN20 represents that the sgRNA transcribed with U6 promoter is 21 nt long and contains an additional G at the 5 'end.
    [00106] The results of the T7E1 assay show (Figure 8) that WT Cas9 exhibits similar editing efficiency when sgRNA transcribed with different strategies was used. However, the editing efficiencies of eSpCas9 (1.1) and SpCas9-HF1 have been significantly reduced when the sgRNA contains an additional nucleotide at the 5 'end. And when using tRNA-sgRNA fusions, the editing efficiencies of eSpCas9 (1.1) and SpCas9-HF1 have been increased to those of WT Cas9 or even more.
    [00107] Regarding specificity editing, WT Cas9 resulted in off-target editing at both off-alvd and off-target sites
    2. eSpCas9 (1.1) and SpCas9-HF1 did not result in off-target editing when tRNA-sgRNA fusions were used.
    Table 4: target gene and oligonucleotide sequence for construction of sgRNA expression vectors
    sgRNA Target string Oligo-F Oligo-R VEGFA-GN19 GGTGAGTGAGTGTG TGCGTGTGG CACCGGTGAGTGAGTGTG TGCGTG AAACCACGCACACACTCACTCACC VEGFA-GN20 GGTGAGTGAGTGTG TGCGTGTGG CACCGGGTGAGTGAGTGT GTGCGTG AAACCACGCACACACTCACTCACCC VEGFA-tRNA-N 2 o GGTGAGTGAGTGTG TGCGTGTGG CACCGaacaaagcaccagtggt ctagtggtagaatagtaccctgccac ggtacagacccgggttcgattcccg gctggtgcaGGTGAGTGAGT GTGTGCGTG AAACCACGCACACA CTCACTCACCtgcacc agccgggaatcgaacccgg gtctgtaccgtggcagggta ctattctaccactagaccactggtgctttgttC
    Petition 870190079766, of 16/08/2019, p. 44/110
    权利要求:
    Claims (15)
    [1]
    1. Genome editing system for site-directed modification of a target sequence in the genome of a cell characterized by the fact that it comprises at least one selected from i) to iii) which follow:
    i) a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion;
    ii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a tRNA-guide RNA fusion; and iii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a tRNA-RNA guide fusion;
    where the Cas9 nuclease variant has greater specificity compared to the wild type Cas9 nuclease, where the 5 'end of the guide RNA is linked to the 3' end of the tRNA, where the fusion is cleaved at the 5 'end of the RNA -guide after being transcribed into the cell, thereby forming a guide RNA that does not carry extra nucleotide at the 5 'end.
    [2]
    2. Genome editing system for site-directed modification of a target sequence in the genome of a cell characterized by the fact that it comprises at least one selected from i) to iii) which follow:
    i) a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion;
    Petition 870190079766, of 16/08/2019, p. 45/110
    2/4 ii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and an expression construct comprising a nucleotide sequence encoding a ribozyme-guide RNA fusion; and iii) an expression construct comprising a nucleotide sequence encoding a Cas9 nuclease variant and a nucleotide sequence encoding a guide ribozyme RNA fusion;
    where the Cas9 nuclease variant has greater specificity compared to the wild type Cas9 nuclease, where the 5 'end of the guide RNA is linked to the 3' end of a first ribozyme, where the first ribozyme is designed to cleave the fusion at the 5 'end of the guide RNA, thereby forming an RNA guide that does not carry extra nucleotide at the 5' end.
    [3]
    3. System, according to claim 1, characterized by the fact that the tRNA and the cell to be modified are derived from the same species.
    [4]
    4. System according to claim 1, characterized by the fact that the tRNA is encoded by a sequence as shown in SEQ ID NO: 1.
    [5]
    5. System according to claim 1 or 2, characterized in that the nuclease variant Cas9 is a variant of SEQ ID NO: 2 and comprises an amino acid substitution at position 855 of SEQ ID NO: 2, for example , the amino acid substitution is K855A.
    [6]
    6. System according to claim 1 or 2, characterized in that the nuclease variant Cas9 is a variant of SEQ ID NO: 2 and comprises amino acid substitutions at positions 810, 1003 and 1060 of SEQ ID NO: 2 , for example, substitutions
    Petition 870190079766, of 16/08/2019, p. 46/110
    3/4 amino acid sections are K810A, K1003A and R1060A.
    [7]
    7. System according to claim 1 or 2, characterized in that the nuclease variant Cas9 is a variant of SEQ ID NO: 2 and comprises amino acid substitutions in positions 848, 1003 and 1060 of SEQ ID NO: 2 , for example, amino acid substitutions are K848A, K1003A and R1060A.
    [8]
    8. System according to claim 1 or 2, characterized in that the nuclease variant Cas9 is a variant of SEQ ID NO: 2 and comprises amino acid substitutions at positions 611, 695 and 926 of SEQ ID NO: 2 , for example, amino acid substitutions are R611 A, Q695A and Q926A.
    [9]
    9. System according to claim 1 or 2, characterized in that the nuclease variant Cas9 is a variant of SEQ ID NO: 2 and comprises amino acid substitutions in positions 497, 611,695 and 926 of SEQ ID NO: 2 , for example, amino acid substitutions are N497A, R611 A, Q695A and Q926A.
    [10]
    10. System according to claim 1 or 2, characterized in that the nuclease variant Cas9 comprises an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
    [11]
    11. System according to claim 1 or 2, characterized by the fact that the nucleotide sequence encoding the Cas9 nuclease variant is optimized at the codon for the organism from which the cell to be modified is derived.
    [12]
    12. System according to claim 1 or 2, characterized by the fact that the guide RNA is a simple guide RNA (sgRNA).
    [13]
    13. Method for genetically modifying a cell characterized by the fact that it comprises: introduction of the system as defined in any one of claims 1 to 12 in the cell and in this way the nuclease variant Cas9 is directed to the target sequence
    Petition 870190079766, of 16/08/2019, p. 47/110
    4/4 in the cell genome by the guide RNA, and results in replacement, deletion and / or addition of one or more nucleotides in the target sequence.
    [14]
    14. Method according to claim 13, characterized in that the cell is derived from mammals such as human, mouse, rat, monkey, dog, pig, sheep, cow and cat; birds such as chicken, duck and goose; plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts and Arabidopsis thaliana.
    [15]
    15. Method according to any one of claims 13 to 14, characterized by the fact that the system is introduced into the cell using a method selected from: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection , viral infection (such as a baculovirus, a vaccinia virus, an adenovirus or other viruses), particle bombardment, PEG-mediated protoplast transformation or agrobacterium-mediated transformation.
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    法律状态:
    2021-06-01| B11A| Dismissal acc. art.33 of ipl - examination not requested within 36 months of filing|
    2021-08-17| B11Y| Definitive dismissal - extension of time limit for request of examination expired [chapter 11.1.1 patent gazette]|
    2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    CN201710089494|2017-02-20|
    PCT/CN2018/076949|WO2018149418A1|2017-02-20|2018-02-22|Genome editing system and method|
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