Crispr effector system based diagnostics

专利摘要:
A lateral flow diagnostic device and methods of using the same are provided herein. the device comprises a substrate and a first end, the first end comprising a sample loading portion. the first end may further comprise a first region loaded with a detectable linker, a crispr effector system, a detection construct, a first test band comprising a biotin linker and a second test band comprising a capture molecule for the linker detectable. the detection construct may comprise an RNA oligonucleotide having a first molecule, such as fitc, at a first end and a second molecule, such as fam, at a second end. contact of the sample loading portion with a sample causes the sample to flow from the sample loading portion of the substrate to the first and second capture regions, thereby generating a detectable signal, which may be indicative of a state of disease.
公开号:BR112020006757A2
申请号:R112020006757-1
申请日:2018-10-04
公开日:2020-10-06
发明作者:Feng Zhang；Jonathan Gootenberg；Omar Abudayyeh
申请人:The Broad Institute Inc.；Massachusetts Institute Of Technology；President And Fellows Of Harvard College；
IPC主号:

专利说明:

[0001] [0001] This application claims the benefit of Interim Application US 62/568,309 filed October 4, 2017, Interim Application US 62/610,144 filed December 22, 2017, Interim Application US 62/623,529 filed January 29, 2018 and Provisional Application US 62/630,787 filed February 14, 2018. The entire contents of the above-identified applications are incorporated herein by reference. DECLARATION CONCERNING THE RESEARCH SPONSORED BY FEDERAL GOVERNMENT
[0002] [0002] This invention was made with government support under grant numbers MH110049 and HL141201 granted by the National Institutes of Health. The government has certain rights to the invention. TECHNICAL FIELD
[0003] [0003] The material disclosed herein is generally aimed at rapid diagnostics related to the use of CRISPR effector systems. RATIONALE
[0004] [0004] Nucleic acids are a universal signature of biological information. The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity on a portable platform has the potential to revolutionize the diagnosis and monitoring of many diseases, provide valuable epidemiological information, and serve as a generalizable scientific tool.
[0005] [0005] In one aspect, the invention provides a lateral flow device comprising a substrate. The substrate may comprise a first end, wherein the first end comprises a sample loading portion. The first end may further comprise a first region loaded with a detectable ligand, a CRISPR effector system, a detection construct, a first test band comprising a biotin ligand and a second test band comprising a capture molecule for the ligand. detectable. The detection construct may comprise an RNA oligonucleotide having a first molecule at a first end and a second molecule at a second end. In certain embodiments, the first molecule can be FITC and the second molecule can be FAM.
[0006] [0006] The lateral flow device may further comprise a cleavable reporter construct comprising a first molecule and a second molecule linked by an RNA or DNA linker. In some embodiments, the first molecule can be FITC and the second molecule can be biotin, or vice versa. The lateral flow device may further comprise a first capture region, which, in some embodiments, may be a first horizontal line through the device. In specific embodiments, the first capture region is proximate and at the same end of the lateral flow substrate as the sample loading portion and may comprise a first binding agent that specifically binds the first molecule of the reporter construct. In some embodiments, the first binding agent can be an antibody, such as an anti-FITC antibody, for example, that is fixed or otherwise immobilized in the first capture region. The lateral flow device may further comprise a second capture region, which, in some embodiments, is located toward the opposite end of the lateral flow substrate from the first binding region. In specific embodiments, the second capture region may comprise a second binding agent that specifically binds to the second molecule of the reporter construct. In some embodiments, the second binding agent can be an antibody, such as an antibiotin antibody, for example, that is fixed or otherwise immobilized in the second capture region.
[0007] [0007] In some embodiments, the detectable ligand can be a gold nanoparticle, which can be modified with a first antibody. In specific embodiments, the first antibody may be an anti-FITC antibody. In some embodiments, the CRISPR effector system may comprise a CRISPR effector protein and one or more guide sequences configured to bind to one or more target sequences.
[0008] [0008] In some embodiments, the substrate may be a substrate of flexible materials, such as a paper substrate or a polymer-based flexible substrate, for example.
[0009] [0009] In certain exemplary embodiments, the CRISPR effector protein may be an RNA-targeted effector protein. In certain embodiments, the RNA targeted to the effector protein may be a Casl3. In specific embodiments, Casl3 can be within 20 kb of a Cas l gene. The Casl13 effector protein can be an organism of a genus selected from the group consisting of: Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifator, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivolaum, Flavobacterium, Flavobivium, Flavobacterium, Flavobacterium Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter and Lachnospira. In specific embodiments, the C2c2 or Casl3b effector protein may be from an organism selected from the group consisting of: Leptotrichia shahii; Leptotrichia wadei (Lw2); Listeria seeligeri; Lachnospiraceae bacterium MAZ020; Lachnospiraceae bacterium NK4A179; [Clostridium] aminophilum DSM 10710; Carnobacterium gallinarum DSM 4847;
[0010] [0010] In certain example embodiments, the CRISPR-Cas effector protein may be a Casl2 protein, such as Cpfl or C2cl.
[0011] [0011] In certain exemplary embodiments, the assay or device may comprise multiple Cas 13 orthologs, multiple Casl2 orthologs, or a combination of Casl2 orthologs.
[0012] [0012] The one or more chi sequences can comprise one or more guide RNAs, which can be designed to bind to one or more target molecules that are diagnostic for a disease state. These disease states may include, but are not limited to, cancer, autoimmune diseases, infections, organ diseases, blood disorders, immune system disorders, brain and nervous system disorders, endocrine disorders, pregnancy-related or childbirth, hereditary diseases or diseases acquired in the environment.
[0013] [0013] In some embodiments, the disease state may be characterized by the presence or absence of a gene or transcript or polypeptide or antibiotic or drug resistance, or preferably in a pathogen or cell.
[0014] [0014] In some embodiments, the infection may be caused by a virus, a bacterium, a fungus, a protozoan or a parasite. In modalities where the infection is viral, it may be caused by a DNA virus. In specific embodiments, the DNA virus may include, but is not necessarily limited to, members of the Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus and Varicella Zoster virus), Malocoherpesviridae, Lipothrixviridae,
[0015] [0015] A viral infection can also be caused by a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, a retrovirus, or a combination thereof. A viral infection may also be caused by a Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, a Orthomyxoviridae, or a Deltavirus. A viral infection can also be caused by Coronavirus, SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue virus, Zika virus, Rubella virus, Ross virus
[0016] [0016] In other embodiments, the infection may be bacterial in nature. The bacterium causing the bacterial infection may include, but is not necessarily limited to, Acinetobacter, a species of Actinobacillus, a species of Actinomycetes, a species of Actinomyces, a species of Aerococcus, a species of Aeromonas, a species of Anaplasma, a species of Alcaligenes , a species of Bacillus, a species of Bacteriodes, a species of Bartonella, a species of Bifidobacterium, a species of Bordetella, a species of Borrelia, a species of Brucella, a species of Burkholderia, a species of Campylobacter, a species of Capnocytophaga , a species of Chlamydia, a species of Citrobacter, a species of Coxiella, a species of Corynbacterium, a species of Clostridium, a species of EFikenella, a species of Enterobacter, a species of Escherichia, a species of Enterococcus, a species of £ Ehlichia, a species of Epidermophyton, a species of Erysipelothrix, a species of Eubacterium, a species of Francisella, a species of Fusob acterium, a species of Gardnerella, a species of Gemella, a species of Haemophilus, a species of Helicobacter, a species of Kingella, a species of Klebsiella, a species of Lactobacillus, a species of Lactococcus, a species of Listeria, a species of Leptospira, a species of Legionella, a species of Leptospira, a species of Leuconostoc, a species of Mannheimia, a species of Microsporum, a species of Micrococcus, a species of Moraxella, a species of Morganell, a species of Mobiluncus, a species of Micrococcus, a species of Mycobacterium, a species of Mycoplasm, a species of Nocardia, a species of Neisseria, a species of Pasteurelaa, a species of Pediococcus, a species of Peptostreptococcus, a species of Pityrosporum, a species of Plesiomonas, a species of Prevotella, a species of Porphyromonas, a species of Proteus, a species of Providencia, a species of Pseudomonas, a species of Propionibacteriums, a species of Rhodo coccus, a species of Rickettsia, a species of Rhodococcus, a species of Serratia, a species of Stenotrophomonas, a species of Salmonella, a species of Serratia, a species of Shigella, a species of Staphylococcus, a species of Streptococcus, a species of Spirillum,y a species of Streptobacillus, a species of Treponema, a species of Tropheryma, a species of Trichophyton, a species of Ureaplasma, a species of Veillonella, a species of Vibrio, a species of Yersinia, a species of Xanthomonas, or combinations of the same.
[0017] [0017] In other embodiments, the infection may be fungal and may be caused by fungi such as, but not necessarily limited to, Aspergillus, Blastomyces, Candidiasis, Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp. Histoplasma sp. (as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), Stachybotrys (such as Stachybotrys chartarum), Mucroymcosis, Sporothrix, mycosis fungal eye infections, Exserohilum, Cladosporium, Geotrichum, Saccharomyces, a species of Hansenula, a species of Candida, a species of Kluyveromyces, a species of Debaryomyces, a species of Pichia, a species of Penicillium,y a species of Cladosporium, a species of Byssochlamys or a combination thereof.
[0018] [0018] In other embodiments, the infection may be caused by a protozoan such as a Euglenozoa, a Heterolobosea, a Diplomonadida, an Amoebozoa, a Blastocystic, an Apicomplexa, or combinations thereof.
[0019] [0019] In other embodiments, the infection can be caused by a parasite such as, but not necessarily limited to, Trypanosoma cruzi (Chagas disease), T. brucei gambiense, T. brucei rhodesiense, Leishmania braziliensis, L. infantun, L. mexicana, L. major, L. tropica, L. donovani, Naegleria fowleri, Giardia intestinalis (G. lamblia, G. duodenalis), canthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica, Blastocystic hominis, Babesia microti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and Toxoplasma gondii, or a combination thereof.
[0020] [0020] In some embodiments, the sample may be a biological sample or an environmental sample. Biological samples may include, but are not necessarily limited to, blood, plasma, serum, urine, feces, sputum, mucosa, lymphatic fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor. , or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation) or fluid obtained from a joint (for example, a normal joint or a joint affected by diseases such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis) or a swab from the surface of the skin or mucous membrane.
[0021] [0021] In certain embodiments, environmental samples may include, but are not necessarily limited to, samples obtained from a food sample, paper surface, fabric, metal surface, wood surface, plastic surface, soil sample, freshwater sample, a waste water sample, a saline water sample, or a combination thereof.
[0022] [0022] In another aspect, the invention provides a lateral flow device comprising a substrate having a first end, wherein the first end comprises a sample loading portion and a first region loaded with a detectable ligand, two or more effector systems. CRISPR, two or more detection constructs, one or more first capture regions, each comprising a first binding agent, two or more second capture regions, each comprising a second binding agent, wherein each of two or more more CRISPR effector systems comprise a CRISPR effector protein and one or more guide sequences, each guide sequence configured to bind one or more target molecules.
[0023] [0023] In some embodiments, each of the two or more detection constructs comprises an RNA or DNA oligonucleotide, comprising a first molecule at a first end and a second molecule at a second end. In specific embodiments, the lateral flow device may comprise two effector systems
[0024] [0024] The sample loading portion may further comprise one or more amplification reagents to amplify the one or more target molecules.
[0025] [0025] In some embodiments, a first detection construct comprises FAM as a second molecule and biotin as a second molecule or vice versa and a second detection construct comprises FAM as a first molecule and Digoxigenin (DIG) as a second molecule or vice versa. In some embodiments, the CRISPR effector protein is an RNA-targeted effector protein. In some embodiments, the RNA-targeting effector protein is C2c2. In some embodiments, the RNA targeting effector protein is Casl3b.
[0026] [0026] In some embodiments, a first detection construct may comprise Tye665 as a first molecule and Alexa-fluor-488 as a second molecule or vice versa; a second detection construct may comprise Tye665 as a first molecule and FAM as a second molecule or vice versa; a third detection construct may comprise Tye665 as a first molecule and biotin as a second molecule or vice versa; and a fourth detection construct may comprise Tye665 as a first molecule and DIG as a second molecule or vice versa.
[0027] [0027] In some embodiments, the CRISPR effector protein may be an RNA-targeted or a DNA-targeted effector protein. The RNA targeting effector can be C2c2 or Casl3b. In some embodiments, the DNA-targeting effector protein is Casl2a.
[0028] [0028] In another aspect, the invention provides a method for detecting a target nucleic acid in a sample, comprising contacting a sample with the first end of the lateral flow device described herein. Preferably, the sample is contacted with the sample loading portion of the device and the sample flows from the sample loading portion of the substrate to the first and second capture regions, thereby generating a detectable signal.
[0029] [0029] The sample can be a liquid sample or it can be dissolved in an aqueous solvent. In embodiments where the sample does not contain target nucleic acid, the detectable signal appears in the first capture region. In embodiments where the sample contains target nucleic acid, the detectable signal appears in the second capture region. The presence of target nucleic acid is typically indicative of a disease state.
[0030] [0030] These and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those skilled in the art by considering the following detailed description of the illustrated exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] [0031] FIG. 1 - is a schematic of an example C2c2 based CRISPR effector system.
[0032] [0032] FIG. 2 - provides (A) the schema of the CRISPR/C2c2 locus of Leptotrichia wadei. Representative crRNA structures of the LwC2c2 and LshC2c2 systems are shown. (SEQ. ID Nos. 220 and 221) (B) Scheme of in vivo bacterial assay for C2c2 activity. A protospacer is cloned upstream of the beta-lactamase gene into an ampicillin-resistant plasmid, and this construct is transformed into E. coli expressing C2c2 together with a targeting or non-targeting spacer. Successful transforms are counted to quantify activity. (C) Quantification of LwC2c2 and Lshc2c2 in vivo. (n = 2 biological replicates; bars represent mean + s.e.m.) (D) Final size exclusion gel filtration of LwCc2c2. (E) Coomassie blue stained acrylamide gel from stepwise purification of LwC2c2. (F) LwC2c2 activity against different PFS targets. LwC2cC2 was directed against fluorescent RNA with
[0033] [0033] FIG. 3 - Shows detection of an example masking construct at different dilutions using 1 ug, 100 ng, 10 ng and 1 ng of target with 4 different amounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 groups of crRNAs, without crRNA condition, technical duplicates, in (96+48)*2 = 288 reactions, measured at an interval of 5 minutes for 3 hours.
[0034] [0034] FIG. 4 - Shows detection of an example masking construct at different dilutions using 1 ug, 100 ng, 10 ng and 1 ng of target with 4 different amounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 groups of crRNAs, without crRNA condition, technical duplicates, in (96+48)*2 = 288 reactions, measured at an interval of 5 minutes for 3 hours.
[0035] [0035] FIG. 5 - Shows detection of an example masking construct at different dilutions using 1 ug, 100 ng, 10 ng and 1 ng of target with 4 different amounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 groups of crRNAs, without crRNA condition, technical duplicates, in (96+48)*2 = 288 reactions, measured at an interval of 5 minutes for 3 hours.
[0036] [0036] FIG. 6 - Shows detection of an example masking construct at different dilutions using 1 ug, 100 ng, 10 ng and 1 ng of target with 4 different amounts of protein/crRNA (1:4, 1:16, 1:32, 1:64) with 2 groups of crRNAs, without crRNA condition, technical duplicates, in (96+48)*2 = 288 reactions, measured at an interval of 5 minutes for 3 hours.
[0037] [0037] FIG. 7 - provides a schematic of an example detection scheme using a masking construct and CRISPR effector protein, according to certain example embodiments.
[0038] [0038] FIG. 8 - provides a set of graphs that show changes in fluorescence over time when detecting a target using different sets of guide RNAs.
[0039] [0039] FIG. 9 - provides a graph showing normalized fluorescence detected at different dilutions of target RNA at varying concentrations of CRISPR effector protein.
[0040] [0040] FIG. 10 - is a schematic showing the general steps of a NASBA amplification reaction.
[0041] [0041] FIG. 11 - provides a graph showing SsRNA 1 detection of NASBA-amplified nucleic acid with three different primer sets and then subjected to collateral detection of C2c2 using a quenched fluorescent probe (n = 2 technical replicates; bars represent mean t without).
[0042] [0042] FIG. 12 - provides a graph showing that the side effect can be used to detect the presence of a lentiviral target RNA.
[0043] [0043] FIG. 13 - provides a graph demonstrating that side effect and NASBA can detect species at concentrations aM.
[0044] [0044] FIG. 14 - provides a graph demonstrating that side effect and NASBA rapidly discriminate low concentration samples.
[0045] [0045] FIG. 15 - Shows that the normalized fluorescence at certain times is predictive of the input concentration of the sample. Fluorescence measurements from Casl3a detection without amplification are correlated with input RNA concentration (n = 2 biological replicates; bars represent mean t+ s.e.m.).
[0046] [0046] FIG. 16 - provides a schematic of the RPA reaction, showing the components participating in the reaction.
[0047] [0047] FIG. 17 - SHERLOCK scheme; provides a schematic showing detection of DNA or RNA targets via incorporation of an RPA or RT-RPA step accordingly. After recognition of the target RNA, the side effect causes C2c2 to cut the cleavage reporter, generating fluorescence. Single-molecule amounts of RNA or DNA can be amplified to DNA via recombinase polymerase (RPA) amplification and transcribed to produce RNA, which is then detected by C2c2.
[0048] [0048] FIG. 18 - provides a schematic of the SSRNA target detected by collateral detection of C2c2 (SEQ. I.D. Nos. 222 and 223).
[0049] [0049] FIG. 19 - provides a set of graphs demonstrating detection of single molecule DNA using RPA (ie 15 minutes after the addition of C2c2).
[0050] [0050] FIG. 20 - provides a set of graphs demonstrating that mixing of T7 polymerase in an RPA reaction adversely affects DNA detection.
[0051] [0051] FIG. 21 - provides a set of graphs demonstrating that mixing the polymerase in an RPA reaction does not adversely affect DNA detection.
[0052] [0052] FIG. 22 - provides a graph demonstrating that RPA, T7 transcription, and C2c2 detection reactions are compatible and achieve single-molecule detection when incubated simultaneously (n = 2 technical replicates; bars represent mean + s.e.m.).
[0053] [0053] FIG. 23 - provides a set of graphs demonstrating the effectiveness of rapid time incubations of RPA-RNA.
[0054] [0054] FIG. 24 - provides a set of graphs demonstrating that increasing the amount of T7 polymerase increases the sensitivity to RNA-RPA.
[0055] [0055] FIG. 25- provides a set of graphs showing the results of an RPA-DNA detection assay using a 1.5x one-pot enzyme reaction. Single molecule detection (2aM) achieved in 30 minutes.
[0056] [0056] FIG. 26 - provides a set of graphs demonstrating that an RPA-DNA potentiometer reaction demonstrates a quantitative decrease in fluorescence relative to the input concentration. The fitted curve reveals relationship between input target concentration and output fluorescence.
[0057] [0057] FIG. 27 - provides a set of graphs demonstrating that (A) detection of C2c2 from RNA without amplification can detect the target SSsSRNA at concentrations below 50 fM (n = 2 technical replicates; bars represent mean + wk) and that (B) the RPA-C2c2 reaction is capable of detecting single-molecule DNA (n = 4 technical replicates; bars represent mean t without).
[0058] [0058] FIG. 28 - provides a set of graphs demonstrating that a C2c2 signal generated according to certain exemplary embodiments can detect a 20 pM target on a paper substrate.
[0059] [0059] FIG. 29 - provides a graph showing that a specific RNAse inhibitor is able to remove the background signal on paper.
[0060] [0060] FIG. 30 is a set of graphs showing detection using systems in accordance with certain exemplary embodiments on fiberglass substrates.
[0061] [0061] FIG. 31 - provides a set of graphs that provide (A) a schematic of Zika RNA detection according to certain example modalities. The lentivirus was packaged with Zika RNA or homologous fragments of dengue RNA, the target of collateral detection of C2c2. The medium is harvested after 48 hours and subjected to thermal lysis, RT-RPA and C2c2 detection. (B) Detection of RT-RAP-C2c2 is capable of highly sensitive detection of lentiviral Zika particles (n = 4 technical replicates, two-tailed Student's t test; t***t**, p <0.0001; bars represent mean t+ w/w) (C) A scheme of Zika RNA detection using lyophilized C2c2 on paper, according to certain example modalities. (D) The paper assay is capable of highly sensitive detection of lentiviral Zika particles (n-4 technical replicates, two-tailed Student's t test; ****, p<0.0001; **, p<0.01 , bars represent mean t+ wk).
[0062] [0062] FIG. 32 - provides a set of graphs demonstrating (A) a scheme for the detection of C2c2 from Zika RNA isolated from human serum. Serum Zika RNA undergoes reverse transcription, RNA RNase H degradation, cCDNA RPA, and C2c2 detection. (B) C2c2 is capable of highly sensitive detection of human serum samples of Zika. The Zika RNA concentrations shown were verified by qPCR (n = 4 technical replicates, two-tailed Student's t test; ****, p <0.0001; bars represent mean + s.e.m.).
[0063] [0063] FIG. 33 - provides a set of graphs demonstrating that (A) lyophilized C2c2 is able to sensitively detect ssRNA 1 in the low femtomolar range. C2c2 is able to rapidly detect a 200 pM SssRNA 1 target on paper in liquid (B) or lyophilized (C) form. The reaction is capable of sensitive detection of Zika RNA fragments synthesized in solution (D) (n = 3) and in lyophilized form (E) (n = 3). (F) Quantitative curve for detection of human Zika cDNA showing significant correlation between input concentration and detected fluorescence. (G) Detection of C2c2 of ssRNA 1 performed in the presence of varying amounts of human serum (n = 2 technical replicates, unless otherwise indicated; bars represent mean t s.e.m.).
[0064] [0064] FIG. 34 - provides (A) the scheme of C2c2 detection of the 16S rRNA gene from bacterial genomes using a universal V3 RPA primer set and (B) the ability to achieve sensitive and specific detection of E. coli or P. aeruginosa gDNA using an assay performed according to certain exemplary embodiments (n = 4 technical replicates, two-tailed Student's t-test; ****, p<0.0001; bars represent mean t+ wk). Ec, Escherichia coli;
[0065] [0065] FIG. 35 - provides a set of graphs demonstrating (A) the detection of two different carbapenem resistance genes (KPC and NDM-1) from four different clinical isolates of Klebsiella pneumoniae and (B) the detection of carbapenem resistance genes (part A) is normalized as a signal ratio between the KPC and NDM-1 crRNA assays (n = 2 technical replicates, two-tailed Student's t test; ****, p < 0.0001; bars represent mean + wk).
[0066] [0066] FIG. 36 - provides a set of graphs demonstrating that (A) c2c2 is not sensitive to single mismatches, but can distinguish between single nucleotide differences in target when loaded with crRNAs with additional mismatches. SSRNA 1-3 targets were detected with 11 crRNAs, with 10 spacers containing —synthetic mismatches at various positions on the crRNA. Mismatched spacers did not show reduced cleavage of target 1, but showed inhibited cleavage of mismatch targets 2 and 3 (SEQ. I.D. Nos. 224 to 237). (B) Schematic showing the process for rational design of specific single-base spacers with synthetic incompatibilities. Synthetic incompatibilities are placed close to the SNP or base of interest (SEQ. I.D. Nos. 238 to 242). (C) Highly specific detection of strain SNPs allows differentiation of African Zika versus American RNA targets that differ by only one nucleotide using detection of C2c2 with truncated crRNAs (23 nucleotides) (n = 2 replicate techniques, t-test of Student with one tail; *, p <0.05; ****, p <0.0001; bars represent mean t+ w/w).
[0067] [0067] FIG. 37 - provides a set of graphs demonstrating: (A) the schematic of the Zika strain target regions and the crRNA sequences used for detection (SEQ. I.D. Nos. 243 to 248). SNPs in the target are highlighted in red or blue and synthetic mismatches in the guide sequence are colored in red. (B) Highly specific detection of strain SNPs allows differentiation of African Zika versus American RNA targets using SHERLOCK (n = 2 replicate techniques, two-tailed Student's t test; ****, p < 0.0001; bars represent mean + wk) (SEQ ID Nos. 249 to 254). (C) Schematic of Dengue strain target regions and crRNA sequences used for detection. SNPs in the target are highlighted in red or blue and synthetic mismatches in the guide sequence are colored in red. (B) Highly specific detection of strain SNPs allows differentiation of Dengue 1 strain targets versus RNA strain
[0068] [0068] FIG. 38 - provides a set of graphs showing (A) circuses plot showing the location of human SNPs detected with C2c2. (B) Assay performed according to certain exemplary modalities can distinguish between human SNPs. SHERLOCK can correctly genotype four different individuals at four different SNP sites in the human genome. The genotypes for each individual and the identities of allele-detecting crRNAs are noted below each graph (n = 4 technical replicates; two-tailed Student's t test; *, p < 0.05; **, p < 0.01; ARA, p <0.001; ARAAA, Pp <0.0001; bars represent mean t+ w/w). (C) An outline of the process for detecting cfDNA (such as detecting cell-free DNA of cancer mutations) according to certain exemplary embodiments. (D) Example of crRNA sequences for detecting EGFR L858R and BRAF V600E (SEQ. I.D. Nos. 255 to 260). Sequences from two genomic loci tested for cancer mutations in cell-free DNA. Shown is the target genomic sequence with the SNP highlighted in blue and the mutant/wild-type detection crRNA sequences with synthetic mismatches colored in red.
[0069] [0069] FIG. 39 - provides a set of graphs demonstrating that C2c2 can detect the mutant minor allele in simulated cell-free DNA samples of the EGFR L858R (C) or the BRAF V600E minor allele (B) (n = 4 technical replicates, Student's t test two-tailed; *, p<0.05; **, p<0.01, ****, P<0.0001; bars represent t without).
[0070] [0070] FIG. 40 - provides a set of graphs demonstrating that (A) the assay can distinguish between genotypes at rs5082 (n = 4 technical replicates; *, p <0.05; **%, p 0.01; ***, p 0.001 ; ** **, p 0.0001; bars represent mean + without). (B) the assay can distinguish between genotypes at rs601338 in gDNA directly from centrifuged, denatured, and boiled saliva (n = 3 technical replicates; *, p < 0.05; bars represent mean + s.e.m.).
[0071] [0071] FIG. 41 - provides (A) a schematic of an example embodiment performed on ssDNA 1 against the background of a target that differs from ssDNA 1 by only a single mismatch. (B) The assay achieves detection of single nucleotide specificity of ssDNA 1 in the presence of a background mismatch (target that differs only by a single ssDNA mismatch). Various concentrations of target DNA were combined with an excess of background DNA with a mismatch and detected by the assay.
[0072] [0072] FIG. 42 is a graph showing a masking construct with a different Cy5 dye also allows for effective detection.
[0073] [0073] FIG. 43 is a schematic of an assay based on colorimetry of gold nanoparticles. AuNPs are aggregated using a combination of DNA ligands and an RNA bridge. Upon addition of RNase activity, the SSRNA bridge is cleaved and AuNPs are released, causing a characteristic color change towards red.
[0074] [0074] FIG. 44 is a graph showing the ability to detect the change in color of dispersed nanoparticles at 520 nm. The nanoparticles were based on the example embodiment shown in Figure 43 and dispersed using the addition of RNase A at varying concentrations.
[0075] [0075] FIG. 45 is a graph showing that the RNase colorimetric test is quantitative.
[0076] [0076] FIG. 46 is an image of a microwell plate showing that the color change in the dispersed nanoparticle is visually detectable.
[0077] [0077] FIG. 47 is a figure demonstrating that colorimetric shift is visible on a paper substrate. The test was performed for 10 minutes at 37 degrees C on 934-AH fiberglass.
[0078] [0078] FIG. 48 is a schematic of conformational switch aptamers according to certain exemplary embodiments for detecting proteins or small molecules. (A) SEQ ID NO:261. Bound product (B) is used as a complete target for the RNA targeting effector, which cannot detect unbound input product (SEQ ID NO: 262).
[0079] [0079] FIG. 49 is an image of a gel showing that aptamer-based binding can create RPA detectable substrates. Aptamers were incubated with various levels of thrombin and then ligated with probe. Bound constructs were used as templates for a 3-minute RPA reaction. Thrombin 500 nM has significantly higher levels of amplified target than background.
[0080] [0080] FIG. 50 shows the amino acid sequence of the HEPN domains of selected C2c2 orthologs (SEQ ID NO: 263-292).
[0081] [0081] FIG. 51 Casl3a RNA detection with RPA amplification (SHERLOCK) can detect the SSRNA target at concentrations up to -2 aM, more sensitive than Casl3a alone (n = 4 technical replicates; bars represent mean + s.e.m.).
[0082] [0082] FIG. 52 - Casl3a detection can be used to detect viral and bacterial pathogens. (A) Schematic of SHERLOCK detection of ZIKV RNA isolated from human clinical samples. (B) SHERLOCK is capable of detecting highly sensitive serum (S) or urine (U) samples positive for human ZIKV. The approximate concentrations of ZIKV RNA shown were determined by qPCR (n = 4 technical replicates, two-tailed Student's t test; ****, p <0.000l1; bars represent mean + s.e.m.; n.d., not detected).
[0083] [0083] FIG. 53 - Comparison of NASBA detection of ssRNA 1 with primer set 2 (from Figure 11) and SHERLOCK (n = 2 technical replicates; bars represent mean t+ s.e.m.).
[0084] [0084] FIG. 54 Single Reaction RPA and SHERLOCK Nucleic Acid Amplification. (A) PCR quantification of ssRNA 1 digital droplets for dilutions used in Fig. 1C. Adjusted concentrations for the dilutions based on the ddPCR results are shown above the bar graphs. (B) PCR quantification of ssDNA 1 digital droplets for dilutions used in Fig. 1D. Adjusted concentrations for the dilutions based on the ddPCR results are shown above the bar graphs. (C) RPA, T7 transcription and Casl3a detection reactions are compatible and achieve detection of single DNA molecule 2 when incubated simultaneously (n = 3 technical replicates, two-tailed Student's t test; ns, not significant; **, p <0.01; ****, p <0.0001; bars represent mean t without).
[0085] [0085] FIG. 55 - Comparison of SHERLOCK with other sensitive nucleic acid detection tools. (THE)
[0086] [0086] FIG. 56 - Detection of resistance to carbapanem in clinical bacterial isolates. Detection of two different carbapenem resistance genes (KPC and NDM-1) from five clinical isolates of Klebsiella pneumoniae and an E. coli control (n = 4 technical replicates, two-tailed Student's t test; ****, p <0.0001; bars represent mean t without; nd, not detected).
[0087] [0087] FIG. 57 - Characterization of Lwasl3a sensitivity to truncated spacers and unique target sequence mismatches. (A) Sequences of truncated spacer crRNAs used in (B) - (GG). Also shown are sequences of SsSsRNA 1 and 2, which have a single base pair difference highlighted in red. CrRNAs containing synthetic mismatches are displayed with mismatch positions colored red (SEQ ID NO: 293-304). (B) Collateral cleavage activity on SssRNA 1 and 2 for the 28 nt spacer crRNA with synthetic mismatches at positions 1-7 (n = 4 technical replicates; bars represent mean t s.e.m.). (C) Specificity ratios of the crRNA tested in (B). Specificity ratios are calculated as the ratio of on-target RNA collateral cleavage (sSSsRNA 1) to off-target RNA collateral cleavage (SsSSRNA 2) (n = 4 technical replicates; bars represent mean + s.e.m.). (D) Collateral cleavage activity on ssRNA 1 and 2 for the 23 nt spacer CcrRNA with synthetic mismatches at positions 1-7 (n = 4 technical replicates; bars represent mean + s.e.m.). (E) crRNA specificity ratios tested in (D). Specificity ratios are calculated as the ratio of collateral cleavage of on-target RNA (sSsRNA 1) to collateral cleavage of off-target RNA (SSRNA 2) (n = 4 technical replicates; bars represent mean + s.e.m.). (F) Collateral cleavage activity on ssRNA 1 and 2 for the 20 nt spacer crRNA with synthetic mismatches at positions 1-7 (n = 4 technical replicates; bars represent mean tt s.e.m.). (G) Specificity ratios of the crRNA tested in (F). Specificity ratios are calculated as the ratio of collateral cleavage of RNA on target (ssRNA 1) to collateral cleavage of RNA off target (sSsSsRNA 2) (n = 4 technical replicates; bars represent mean t s.e.m.).
[0088] [0088] FIG. 58 - Identification of the ideal position of synthetic mismatch in relation to mutations in the target sequence. (A) Sequences to assess the ideal position of synthetic mismatch to detect a mutation between ssRNA 1 and ssRNA 2. In each of the targets, crRNAs with synthetic mismatches at the colored (red) sites are tested. Each set of synthetic mismatch crRNAs is designed so that the mutation site is shifted in position relative to the spacer sequence. The spacers are designed so that the mutation is evaluated at positions 3, 4, 5 and 6 within the spacer. SEQ ID NOs: 305-336 are shown. (B) Collateral cleavage activity on ssRNA 1 and 2 for crRNAs with synthetic mismatches at varying positions. There are four sets of crRNAs with the mutation at positions 3, 4, 5 or 6 within the spacer: target duplex region (n = 4 technical replicates; bars represent mean + s.e.m.). (C) Specificity ratios of the crRNA tested in (B). Specificity ratios are calculated as the ratio of collateral cleavage of RNA on target (ssRNA 1) to collateral cleavage of RNA off target (sSsSsSRNA 2) (n = 4 technical replicates; bars represent mean + s.e.m.).
[0089] [0089] FIG. 59 - SHERLOCK genotyping at an additional locus and direct genotyping from boiled saliva. SHERLOCK can distinguish between genotypes at the rs601338 SNP site in genomic DNA directly from centrifuged, denatured and boiled saliva (n = 4 technical replicates, two-tailed Student's t test; **, p < 0.01; ****, p < 0.001; bars represent mean t without).
[0090] [0090] FIG. 60 - Development of synthetic genotyping standards for accurate genotyping of human SNPs. (A) SHERLOCK genotyping at the rs601338 SNP site for each of the four subjects compared to PCR amplified genotype patterns (n = 4 technical replicates; bars represent mean + s.e.m.). (B) SHERLOCK genotyping at the rs4363657 SNP site for each of the four individuals compared to PCR amplified genotype patterns (n = 4 technical replicates; bars represent mean t+ s.e.m.). (C) Heat maps of the p-values calculated between the SHERLOCK results for each individual and the synthetic patterns at the rs601338 SNP site. A heat map is shown for each of the allele-detecting CrRNAs.
[0091] [0091] FIG. 61 - Detection of ssDNA 1 as a small fraction of incompatible background target. SHERLOCK detection of a ssDNA 1 dilution series on a human genomic DNA background. Note that there should be no sequence similarity between the ssDNA 1 target being detected and the background genomic DNA (n = 2 technical replicates; bars represent mean t s.e.m.).
[0092] [0092] FIG. 62 - Urine (A) or serum (B) samples from patients with Zika virus were inactivated by heat for 5 minutes at 95º C (urine) or 65º C (serum). One microliter of inactivated urine or serum was used as input for a 2 hour RPA reaction followed by a 3 hour C2c2/Casl3a detection reaction, according to an exemplary embodiment. Error bars indicate 1 SD based on n = 4 technical replicates for the detection reaction.
[0093] [0093] FIG. 63 - Urine samples from patients with Zika virus were heat-inactivated for 5 minutes at 95°C. One microliter of inactivated urine was used as input for a 30-minute RPA reaction, followed by a 3-minute C2c2/Cas13 detection reaction. hours (A) or 1 hour (B), according to example modalities. Error bars indicate 1 SD based on n = 4 technical replicates for the detection reaction.
[0094] [0094] FIG. 64 - Urine samples from patients with Zika virus were inactivated by 5 minutes at 95°C. One microliter of inactivated urine was used as input for a 20 minute RPA reaction followed by a 1 hour C2c2/Casl3a detection reaction. Healthy human urine was used as a negative control. Error bars indicate 1 SD based on n = 4 technical replicates for the detection reaction.
[0095] [0095] FIG. 65 - Urine samples from patients with Zika virus were inactivated by 5 minutes at 95°C. One microliter of inactivated urine was used as input for a 20 minute RPA reaction followed by a 1 hour C2c2/Casl3a detection reaction in the presence or absence of guide RNA. Data are normalized by subtracting the mean fluorescence values for unguided detection reactions from reactions that contain guides. Healthy human urine was used as a negative control. Error bars indicate 1 SD based on n = 4 technical replicates for the detection reaction.
[0096] [0096] FIG. 66 - Shows detection of two malaria-specific targets with four different lead RNA designs, according to example modalities. SEQ ID NOs: 337-348 are shown.
[0097] [0097] FIG. 67 - Provides graphs showing editing preferences of different Casl3b orthologs. See Table 3 for the key.
[0098] [0098] FIG. 68 - provides A) a schematic of a multiplex assay using different Casl3b orthologs with different editing preferences and B) data demonstrating the feasibility of an assay using Casl13b10 and Casl3b5.
[0099] [0099] FIG. 69 - provides graphs showing double multiplexing with Casl13b5 (Prevotella sp. Orthologs MA2106) and Casl3b9 (Prevotella intermedia). Effector proteins and guide sequences were contained in the same reaction, allowing double multiplexing in the same reaction, using different fluorescent readouts (poly U 530 nm and poly A 485 nm).
[0100] [0100] FIG. 70 - provides the same as in FIG. 69 but in this case using Casl3a orthologs (Leptorichia wadei LwaCasl3a) and Casl3b orthologs (Prevotella sp. MA2Z016, Casl13b5).
[0101] [0101] FIG. 71 - provides a method for grouping target sequences with multiple guide sequences in order to determine the robustness of segmentation, in accordance with certain example modalities. Shown are SEQ ID NO: 349 and
[0102] [0102] FIG. 72 - provides hybrid chain reaction (HCR) gels showing that Casl3 effector proteins can be used to unlock a primer, e.g. a primer embedded in a masking construct, as described here, to activate a hybridization chain reaction .
[0103] [0103] FIG. 73 - provides data showing the ability to detect Pseudomonas aeruginosa in complex lysate.
[0104] [0104] FIG. 74 - provides data showing ion preferences of certain Casl3 orthologs according to certain example modalities. All target concentrations were 20 nM input with ion concentrations of (1mM and 10mM).
[0105] [0105] FIG. 75 - provides data showing that Casl13b12 has a preference for ImM zinc sulfate for cleavage.
[0106] [0106] FIG. 76 - provides data showing that tapping optimization can increase the noise signal of Casl3b5 in a polyA reporter. The old buffer comprises 40 mM Tris-HCL, 60 mM NaCl, 6 mM MgCl 2 , pH 7.3. The new buffer comprises 20 mM HEPES, pH 6.8, 6 mM MgCl2 and 60 mM NaCl.
[0107] [0107] FIG. 77 - provides a schematic of the systems
[0108] [0108] FIG. 78 - provides relative cleavage activity at different nucleotides of various orthologs of Casl3b and in relation to an LwCcasl3a.
[0109] [0109] FIG. 79 - provides a graph showing the relative sensitivity of various examples of Cas13 orthologs.
[0110] [0110] FIG. 80 - provides graph showing the ability to achieve zepto molar (ZM) detection levels using an example modality.
[0111] [0111] FIG. 81 - provides the schematic of a multiplex assay using Casl3 orthologs with different editing preferences and polyN-based masking constructs.
[0112] [0112] FIG 82 - provides data showing the results of primer optimization experiments and detection of pseudomonas under a variety of conditions.
[0113] [0113] FIG. 83 - provides a schematic of a lateral flow test and results obtained using a lateral flow device in accordance with certain exemplary embodiments.
[0114] [0114] FIG. 84 - provides the ability to detect specific soybean spots using a lateral flow device in accordance with certain example modalities.
[0115] [0115] FIG. 85 - Adapting the SHERLOCK for lateral flow detection. (A) Schematic of lateral flow detection with SHERLOCK. (B) Detection of Zika synthetic RNA using lateral flow SHERLOCK with 1 hour LwaCasl3a reaction. (C) Quantification of band intensity from detection in (B).
[0116] [0116] FIG. 86 - Agricultural and point-of-care sanitary applications with lateral flow SHERLOCK. (A) Detection of the CP4-EPSPS herbicide resistance gene or lectin control in soybean seeds modified with CP4-EPSPS (herbicide resistant) or wT using LwaCasl3a. (B) Time course of detection of CP4-EPSPS in herbicide resistant soybean or WT. (C) Time course of lectin detection in herbicide resistant soybean or WT. (D) Quantification of CP4-EPSPS DNA content of CP4-EPSPS and WT seed mixtures with LwaCasl3a. (E) Detection of lateral flow of the CP4-EPSPS herbicide resistance gene or lectin control in soybean seeds modified with CP4-EPSPS or WT using LwaCasl3a. (E) Quantification of band intensity from detection in (E). (G) Detection of the EGFR L858R mutation in cell-free DNA samples derived from patients with L858R or WT cancer mutations. (H) Lateral flow detection of the EGFR L858R mutation in cell-free DNA samples derived from patients with L858R or WT cancer mutations. (YOU)
[0117] [0117] FIG. 87 - Lateral magnetic flux based on SHERLOCK spheres. (A) Schematic of lateral flow reading based on SHERLOCK magnetic spheres. Collateral activity separates the lateral flow reporters from the beads, allowing detection. (B) Dengue synthetic RNA detection using magnetic bead-based lateral flow SHERLOCK with 1 hour LwaCasl3a reaction. (C) Quantification of band intensity from detection in (B).
[0118] [0118] FIG. 88 - SHERLOCK lateral flow detection of ssRNAl. (A) Detection of ssRNA 1 using SHERLOCK lateral flow at various concentrations of SSRNA 1. (B) Quantification of band intensity from detection in (A).
[0119] [0119] FIG. 89 - SHERLOCK lateral flow detection of CP4 genes and lectin from crude plant extract. (THE
[0120] [0120] FIG. 90 - Detection of SHERLOCK lateral flow in exon 19 synthetic deletion samples. (A) Detection of EGFR exon 19 deletion mutation in exon 19 deleted synthetic DNA samples or WT genotype using LwaCasl3a. (B) Lateral flow detection of EGFR exon 19 deletion mutation in exon 19 deleted synthetic DNA samples or WTI genotype using LwaCasl3a. (C) Quantification of band intensity from detection in (B).
[0121] [0121] FIG. 91 - illustrates detection of the soybean herbicide resistance gene with SHERLOCK. (A) A SHERLOCK scheme in combination with a rapid method of genomic DNA extraction, which allows detection of soybean transgenes in a quantitative, multiplexed, and portable manner via side flow strips. (B) SHERLOCK detection of the Roundup Ready (RR) CP4 EPSPS transgene and a lectin positive control gene using LwaCasl3a and a fluorescent reporter. (C) Quantitative SHERLOCK detection of the percentage of Roundup Ready (RR) CP4 EPSPS transgene in a complex soybean mixture. (D) Schematic of multiplexed detection in sample of the CP4 EPSPS transgene and lectin using two-color SHERLOCK with LwaCasl3a and PsmCasl13b. (E) Sample-multiplexed detection of CP4 EPSPS transgene and lectin using two-color SHERLOCK with LwaCasl3a and PsmCasl3b. Soy lectin detection is compared to a control sample of water without input. (F) Schematic of rapid detection of soybean transgene using SHERLOCK side flow strips. (G) Rapid detection of the CP4 EPSPS transgene within 30 minutes on lateral flow strips using SHERLOCK and LwaCasl3a. (H) Quantification of the sample band intensities of the lateral flow strips in G.
[0122] [0122] FIG. 92 - illustrates the kinetics of plant gene detection with SHERLOCK. (A) Detection of the Roundup Ready (RR) CP4 EPSPS transgene using SHERLOCK and LwaCasl3a in RR soybean and wild type (WT) soybean over time. (B) Lectin gene detection using SHERLOCK and LwaCasl3a in Roundup Ready (RR) and wild-type (WT) soybeans over time.
[0123] [0123] FIG. 93 - illustrates the quantitative SHERLOCK of the CP4 EPSPS transgene in a soybean population. (THE)
[0124] [0124] FIG. 94 - illustrates SHERLOCK detection of the CP4 EPSPS transgene with Csm6 signal amplification. (A) SHERLOCK detection of the Roundup Ready (RR) CP4 EPSPS transgene with LwaCasl3a and signal amplification with EiCsm6 or LsCsm6. (B) EiCsm6 amplification kinetics of SHERLOCK detection of the CP4 EPSPS transgene with LwaCasl3a.
[0125] [0125] FIG. 95. - provides results of colorimetric detection of RNase activity with aggregation of gold nanoparticles. A) scheme of colorimetric reading based on gold nanoparticles for RNase activity. In the absence of RNase activity, RNA ligands aggregate gold nanoparticles, leading to loss of red color. Cleavage of the RNA ligands releases nanoparticles and results in a red color change.
[0126] [0126] FIG. 96. - A) provides a lateral flow detection scheme, according to certain exemplary embodiments.
[0127] [0127] FIG 97 - A) detection of ssRNA 1 using lateral flow mode at various concentrations. B) Quantification of the band intensity from the detection in (A).
[0128] [0128] FIG. 98. Single-pot lateral flow genotyping of saliva genomic DNA. A) Scheme for rapid extraction and detection of a pot of genomic DNA from the patient's saliva. B). Detection of rs601338 genotypes from raw DNA extraction compared to water input. Ç). Lateral flow detection of rs601338 genotypes from crude genomic DNA extraction. D). Quantification of detection band intensity in (C) E) Detection of patient DNA in 25 minutes from raw saliva
[0129] [0129] FIG. 99 - SHERLOCK lateral flow detection of synthetic cfDNA samples. A) Detection of EGFT exon 19 deletion mutation in exon 19 deleted synthetic DNA samples or WT genotype using LwaCasl3a. B) Lateral flow detection of EGFR exon 19 deletion mutation in exon 19 deleted synthetic DNA samples of WT genotype using LwaCasl3a. C) Quantification of the band intensity from the detection in (B). D)
[0130] [0130] FIG. 100 - SHERLOCK enhanced with lateral flow Csm6 with different reporter combinations. A) Sidestream detection of SHERLOCK enhanced with Csm6 with multiple reporter designs. sA: short pulley sensor; 1A: long polyA sensor; sC: short poly-C sensor; 1C; long poly-C sensor; sA/C: short poly-A/C sensor; 1A/C: long poly-A/C sensor; M: mixed RNase alert sensor. B) Quantification of the band intensity from the detection in (A). C) Schematic of lateral flow readout of EiCsm6 enhanced LwaCasl3a SHERLOCK detection of ssDNA acyltransferase with separate RPA and IVT steps. EiCsm6 enhanced lateral flow SHERLOCK with P. aeruoginosa acyltransferase gene in combination with LwasCasl3a. The quantification of band intensity is shown on the right.
[0131] [0131] FIG. 101 - Effect of crRNA spacer length on Casdl3 ortholog cleavage. A) Cleavage activity of PsmCasl3b with crRNAs targeting sSSRNAl of variable spacer lengths. B) Cleavage activity of CcaCasl3b with crRNAs targeting SSRNAl of variable spacer lengths.
[0132] [0132] FIG. 102 - Relation of Casl3 families with preferences for cleavage by dinucleotides. A) Protein sequence similarity matrix based on alignment of multiple protein sequences from various orthologous members of Casl3a and Casl3b. Clustering is shown based on Euclidean distance. B) Forward repeating sequence similarity matrix based on multiple sequence alignment of various forward repeating sequences of Cas136a and Cas13b. Clustering is shown based on Euclidean distance. C) Pooling of Casl13 cleavage activity base preferences of dinucleotide motif reporters.
[0133] [0133] FIGsS 103A and 103B - Show results of lateral flow assay for dengue RNA and sSsRNAl using a Casl13b10 probe for dengue and a LwaCasl3a probe for SSRNAl.
[0134] [0134] unless stated otherwise, technical and scientific terms used herein have the same meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2" edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4*" edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic
[0135] [0135] As used in this document, the singular forms "a", "an", and "the" include singular and plural referents, unless the context clearly indicates otherwise.
[0136] [0136] the term "optional" or "optionally" means that the event, circumstance or subsequent substituent described may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
[0137] [0137] Recitation of numeric ranges by endpoints includes all numbers and fractions included in the respective ranges, as well as the endpoints recited.
[0138] [0138] The terms "about" or "approximately", as used in this document, when referring to a measurable value, such as a parameter, an amount, a time duration, and the like, are intended to encompass variations to and from of the specified value, as variations of +/- 10% or less, +/- 5% or less, +/- 1% or less, and +/- 0.1% or less of and from the specified value, to the extent that such variations are suitable for carrying out the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers is also specifically and preferably disclosed.
[0139] [0139] Reference throughout this specification to "a modality", "a modality", "an example modality" means that the specific feature, structure or feature described in connection with the modality is included in at least one modality of the modality. present invention. Thus, the appearances of the phrases "in an embodiment", "in an embodiment" or "an example embodiment" in various places throughout this descriptive report are not necessarily all referring to the same embodiment, but they may. Furthermore, the particular features, structures or features may be combined in any suitable manner, as would be apparent to one skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described in this document include some, but not others, features included in other embodiments, combinations of features from different embodiments should be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination.
[0140] [0140] "C2c2" is now referred to as "Casl3a" and the terms are used interchangeably here unless otherwise noted.
[0141] [0141] All publications, published patent documents and patent applications mentioned herein are incorporated by reference to the same extent that each individual publication, published patent document or patent application has been specifically and individually indicated as being incorporated by reference.
[0142] [0142] The modalities disclosed herein utilize RNA or DNA targeting effectors to provide a robust CRISPR-based diagnosis with attomolar sensitivity. Embodiments disclosed herein can detect DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. In addition, the modalities disclosed herein may be prepared in lyophilized format for convenient distribution and point-of-care (POC) applications. Such modalities are useful in a variety of settings in human health, including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of free DNA from disease-associated cells. In certain embodiments, the present invention is used for rapid detection of foodborne pathogens using pathogen-specific guide RNAs (e.g., Campylobacter jejuni, Clostridium perfringens, Salmonella spp., Escherichia coli, Bacillus cereus, Listeria monocytogenes, Shigella spPp. , Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotuberculosis, Brucella sSPpPp., Corynebacterium ulcerans, Coxiella burnetii, or Plesiomonas shigelloides).
[0143] [0143] In one aspect, the modalities disclosed herein are directed to a nucleic acid detection system comprising a CRISPR system, one or more
[0144] [0144] For ease of reference, these systems may be referred to here as SHERLOCK systems and the reactions they facilitate as SHERLOCK reactions. If a target molecule is present in a sample, the corresponding guide molecule will guide the CRSIPR Cas/guide complex to the target molecule, hybridizing to the target molecule, thereby triggering the nuclease activity of the CRISPR effector protein. This activated CRISPR effector protein will cleave the target molecule and then non-specifically cleave the binding portion of the RNA construct.
[0145] [0145] The modalities disclosed in this document are directed to lateral flow detection devices that comprise SHERLOCK systems. The device may comprise a lateral flow substrate for detecting a SHERLOCK reaction. Substrates suitable for use in lateral flow assays are known in the art. This may include, but is not necessarily limited to, membranes or pads made from cellulose and/or fiberglass, polyesters, nitrocellulose, or absorbent pads (J Saudi Chem Soc 19 (6): 689-705; 2015). The SHERLOCK system, i.e., one or more CRISPR systems and corresponding reporter constructs are added to the side-flow substrate in a defined reagent portion of the side-flow substrate, typically at one end of the side-flow substrate. Reporting constructs used within the context of the present invention comprise a first molecule and a second molecule linked by an RNA or DNA linker.
[0146] [0146] Side support substrates can be located inside a housing (see eg “Rapid Lateral Flow Test Strips” Merck Millipore 2013). The housing may comprise at least one opening for loading samples and a single second opening or separate openings that allow reading of the detectable signal generated in the first and second capture regions.
[0147] [0147] The SHERLOCK system can be lyophilized on the side-flow substrate and packaged as a ready-to-use device, or the SHERLOCK system can be added to the reagent portion of the side-flow substrate at the time of use of the device. The samples to be screened are loaded onto the sample loading portion of the side-flow substrate. Samples should be liquid samples or samples dissolved in an appropriate solvent, usually aqueous. The liquid sample reconstitutes the SHERLOCK reagents so that a reaction
[0148] [0148] Specific binding integrating molecules comprise any members of binding pairs that can be used in the present invention. Such binding pairs are known to those skilled in the art and include, but are not limited to, antibody-antigen pairs, enzyme-substrate pairs, receptor-ligand pairs, and streptavidin-biotin. In addition to these known binding pairs, new binding pairs can be specifically designed. A characteristic of binding pairs is the bonding between the two members of the binding pair.
[0149] [0149] Oligonucleotide linkers that have molecules at each end may comprise DNA if the CRISPR effector protein has DNA collateral activity (Cpfl and C2cl) or RNA if the CRISPR effector protein has RNA collateral activity. oligonucleotide linkers can be single-stranded or double-stranded and, in certain embodiments, they can contain regions of RNA and DNA. Oligonucleotide linkers can be of varying lengths, such as 5-10 nucleotides, 10-20 nucleotides, 20-50 nucleotides, or more.
[0150] [0150] In some embodiments, the polypeptide identifier elements include affinity tags such as hemagglutinin (HA) tags, Myc tags, FLAG tags, V5 tags, chitin binding protein (CBP) tags, binding protein tags maltose (MBP), GST tags, poly-His tags, and fluorescent proteins (e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), dsRed, mCherry, Kaede, Kindling and derivatives thereof, FLAG tags, Myc tags, AUl tags, T7 tags, OLLAS tags, Glu-Glu tags, VSV tags, or a combination thereof. Other affinity tags are well known in the art. Such tags can be detected and/or isolated using methods known in the art (e.g. using specific binding agents such as antibodies that recognize a particular affinity tag). os) may further contain, for example, detectable labels such as isotope labels and/or nucleic acid barcodes such as those described herein.
[0151] [0151] For example, a lateral flow strip allows detection of RNAse (eg Casl3a) by color. The RNA reporter is modified to have a first molecule (such as FITC) attached to the 5' end and a second molecule (such as biotin) attached to the 3' end (or vice versa). The lateral flow strip is designed to have two lines of capture with anti-first molecule antibodies (eg anti-FITC) hybridized in the first line and anti-second molecule antibodies (eg antibiotin) in the second line downstream. As the SHERLOCK reaction flows through the strip, the uncleaved reporter binds to the anti-first molecule antibodies in the first capture line, while the cleaved reporters release the second molecule and allow the second molecule to bind in the second capture line. Second molecule sandwich antibodies, for example conjugated to nanoparticles such as gold nanoparticles, bind any second molecule in the first or second row and result in a strong read/signal (e.g. color). As more reporter is cleaved, more signal accumulates in the second capture line and less signal appears in the first line. In certain aspects, the invention pertains to the use of a strip below, as described herein, for the detection of nucleic acids or polypeptides. In certain aspects, the invention pertains to a method for detecting nucleic acids or polypeptides with a flow strip as defined herein, for example flow (side) tests or flow (side) immunochromatographic assays.
[0152] [0152] In certain exemplary embodiments, a lateral flow device comprises a lateral flow substrate comprising a first end for application of a sample. The first region is loaded with a detectable ligand such as those disclosed herein, for example a gold nanoparticle. The gold nanoparticle can be modified with a first antibody, such as an anti-FITC antibody.
[0153] [0153] CRISPR-associated adaptive immune systems (CRISPR-Cas) contain programmable endonucleases such as Cas9º and Cpfl (Shmakov et al., 2017; Zetsche et al., 2015). Although both Cas9 and Cpfl target DNA, RNA-guided single-effector RNases have recently been discovered (Shmakov et al., 2015) and characterized (Abudayyeh et al., 2016; Smargon et al., 2017), including C2c2, providing a platform for specific RNA detection. RNA-guided RNases can be easily and conveniently reprogrammed using CRISPR RNAs (crRNAs) to cleave the target RNAs. Unlike the Cas9 and Cpfl DNA endonucleases, which only cleave their DNA target, RNA-guided RNases such as C2c2 remain active after cleavage of their RNA target, leading to "collateral" cleavage of non-target RNAs nearby. (Abudayyeh et al., 2016). This crRNA-programmed collateral RNA cleavage activity presents the opportunity to use RNA-guided RNases to detect the presence of a specific RNA, triggering in vivo programmed cell death or in vitro nonspecific degradation of RNA that can serve as a readout (Abudayyeh et al. ., 2016; East-Seletsky et al., 2016). Collateral activity was also recognized in other CRISPR Cas enzymes [rimary flag to give me citations of collateral activity of Cpfl1 and C2cl]. CRISPR EFFECTOR PROTEINS
[0154] [0154] In general, a CRISPR-Cas or CRISPR system, as used in this document and in documents such as WO 2014/093622 (PCT/US2013/074667), collectively refers to transcripts and other elements involved in expression or direction of the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr sequence (trans activating CRISPR) (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (which includes a "forward repeat" and a partial forward repeat processed by tracrRNA in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), or "RNA(s)", such as this term is used here (e.g. RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivator RNA (tracr) or a single guide RNA (S9gRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at sites of a target sequence (also known as a protospacer in the context of an endogenous CRISPR system). When the CRISPR protein is a C2c2 protein, a tracrRNA is not required. C2c2 was described in Abudayyeh et al. (2016) “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”; Science; DOI: 10.1126/science.aaf5573; and Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, DOI: dx.doi.org/10.1016/j.molcel.2015.10.008; which are incorporated herein in their entirety by reference. Casl3b was described in Smargon et al. (2017) “Casl3b Is a Type VI-B CRISPR-Associated RNA-Guided RNases Differentially Regulated by Accessory Proteins Csx27 and Csx28,” Molecular Cell. 65, 1-13; dx.doi.org/10.1016/j.molcel.2016.12.023., which is incorporated herein in its entirety by reference.
[0155] [0155] In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs the binding of the effector protein complex, as disclosed herein, to the target site of interest. In some embodiments, the PAM may be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM may be a 3' PAM (i.e. located downstream of the 5' end of the protospacer). The term "PAM" may be used interchangeably with the term "PFS" or "protospacer flanking site" or "protospacer flanking sequence".
[0156] [0156] In a preferred embodiment, the CRISPR effector protein may recognize a 3' PAM. In certain embodiments, the CRISPR effector protein may recognize a 3' PAM that is 5'H, where H is A, C, or U. In certain embodiments, the effector protein may be Leptotrichia shahii C2c2p, more preferably Leptotrichia shahii DSM 19757 C2c2, and PAM 3' is a 5' H.
[0157] [0157] In the context of forming a CRISPR complex, "target sequence" refers to a sequence in which a leader sequence is designed to have complementarity, where hybridization between a target sequence and a leader sequence promotes the formation of a CRISPR complex. A target sequence may comprise RNA polynucleotides. The term "target RNA" refers to an RNA polynucleotide that is or comprises the target sequence. In other words, the target RNA may be an RNA polynucleotide or a part of an RNA polynucleotide in which a part of the gRNA, i.e., the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising the CRISPR effector protein and a gRNA must be targeted. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.
[0158] [0158] The nucleic acid molecule encoding a CRISPR effector protein, in particular C2c02, is advantageously the codon-optimized CRISPR effector protein. An example of a codon-optimized sequence is, in this case, a sequence optimized for expression in eukaryotes, e.g., humans (i.e., being optimized for expression in humans), or for another eukaryote, animal, or mammal, as discussed herein; see, for example, human codon optimized sequence SaCas9 in WO 2014/093622 (PCT/US2013/074667). While this is preferred, it will be appreciated that other examples are possible and codon optimization for a non-human host species, or codon optimization for specific organs, is known. In some embodiments, an enzyme coding sequence encoding a CRISPR effector protein is a codon optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells can be those of or derived from a specific organism, such as a plant or a mammal, including but not limited to eukaryotes or human or non-human animals or mammals, as discussed herein, for example, mouse, rat, rabbit, dog, cattle or non-human mammal or primate.
[0159] [0159] In certain embodiments, the methods described herein may comprise providing a Cas transgenic cell, in particular a C2c2 transgenic cell, in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced operatively linked into the cell with a regulatory element comprising a promoter from one or more genes of interest. As used herein, the term "Cas transgenic cell" refers to a cell, such as a eukaryotic cell, into which a Cas gene has been genomically integrated.
[0160] [0160] It will be understood by the learned that the cell, such as the transgenic Cas cell, as referred to herein, may comprise other genomic alterations, in addition to having an integrated Cas gene or mutations resulting from the specific action of the Cas sequence when complexed with RNA capable of guide Cas to a locus of destiny.
[0161] [0161] In certain aspects, the invention involves vectors, for example, to deliver or introduce into a cell Cas and/or RNA capable of guiding Cas to a target locus (i.e. guide RNA)Õ3 but also to propagate these components ( e.g. in prokaryotic cells). As used in this document, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, like a plasmid, phage, or cosmid, into which another DNA segment can be inserted, so as to cause the inserted segment to replicate.
[0162] [0162] Recombinant expression vectors may comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected with base on the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that permits expression of the nucleotide sequence (e.g., in a transcription/translation system in vitro or in a host cell when the vector is introduced into the host cell). With respect to methods of recombination and cloning, U.S. Patent Application 10/815,730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety, is mentioned. Thus, the modalities disclosed herein may also comprise transgenic cells comprising the CRISPR effector system. In certain exemplary embodiments, the transgenic cell may function as an individual discrete volume. In other words, samples comprising a masking construct can be delivered to a cell, for example, in a suitable delivery vesicle and if the target is present in the delivery vesicle, the CRISPR effector is activated and a detectable signal is generated.
[0163] [0163] The vectors may include the regulatory element(s), e.g. promoter(s). The vector(s) may comprise Cas and/or a single coding sequences, but possibly also may comprise at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA(s) coding sequences, e.g. sgRNAs such as 1-2, 1-3, 1-4 1-5, 3 -6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48 3-50 RNA(s) (e.g. sogRNAs In a single vector, there may be one promoter for each RNA (eg, SgRNA), advantageously when there are up to about 16 RNA(s); and, when a single vector provides more than 16 RNA(s), one or more more promoters can drive the expression of more than one RNA(s), e.g. when there are 32 RNA(s), each promoter can drive the expression of two RNA(s), and when there are 48 RNA(s), each promoter can direct the expression of three RNA(s).By simple arithmetic and well-established cloning protocols and the teachings of this disclosure, one skilled in the art can and readily practicing the invention as to the RNA(s) for a suitable exemplary vector, such as AAV, and a suitable promoter, such as the U6 promoter. For example, the AAV packaging limit is -4.7 kb.
[0164] [0164] RNA coding sequences (lead(s) and/or Cas coding sequences (cas) may be functionally or operationally linked to regulatory element(s) and therefore the element(s) regulator(s) direct expression. The promoter(s) may be constitutive promoters and/or conditional promoters and/or inducible promoters and/or tissue-specific promoters. The promoter may be selected from the group consisting of RNA polymerases , pol I, pol II, pol III, T/ , U6, HI, Rous sarcoma virus (RSV) retroviral LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the of B-actin, phosphoglycerol kinase (PGK) promoter and EF1a promoter An advantageous promoter is the U6 promoter.
[0165] [0165] In some embodiments, one or more elements of a nucleic acid targeting system is derived from a particular organism that comprises an endogenous CRISPR RNA targeting system. In certain exemplary embodiments, the CRISPR effector protein RNA targeting system comprises at least one HEPN domain, including, but not limited to, the HEPN domains described herein, HEPN domains known in the art, and domains recognized as HEPN domains compared to consensus sequence motifs. Several such domains are provided here. In a non-limiting example, a consensus sequence can be derived from the C2c2 or Casl3b ortholog sequences provided herein. In certain exemplary embodiments, the effector protein comprises a single HEPN domain. In certain other exemplary embodiments, the effector protein comprises two HEPN domains.
[0166] [0166] In an exemplary embodiment, the effector protein comprises one or more HEPN domains comprising an RxxxxH motif sequence. The RxxxxH motif sequence can be, without limitation, from a HEPN domain described herein or a HEPN domain known in the art. The RxxxxH motif sequences further include motif sequences created by combining portions of two or more HEPN domains. As noted, the consensus sequences can be derived from the sequences of the orthologs disclosed in Interim Patent Application US 62/432,240, titled "Novel CRISPR Enzymes and Systems," Interim Patent Application US 62/471,710, titled "Novel Type VI CRISPR Orthologs and Systems” filed March 15, 2017 and US Interim Patent Application titled “Novel Type VI Orthologs and Systems CRISPR,” labeled Attorney File Number 47627-05-2133 and filed April 12, 2017.
[0167] [0167] In one embodiment of the invention, a HEPN domain comprises at least one RxxxxH motif comprising the sequence of R(N/H/K)XIX2X3H (SEQ ID NO:351). In one embodiment of the invention, a HEPN domain comprises an RxxxxH motif comprising the sequence of R(N/H)X1X2X3H (SEQ ID NO:352). In one embodiment of the invention, a HEPN domain comprises the sequence RIN/K)XIX2X3H (SEQ ID NO:353). In certain embodiments, X1 is R, S, D, E, Q, N, G, Y, or H. In certain embodiments, X2 is I, S, T, V, or L. In certain embodiments, X3 is L, F, N, Y, V, I, S, D, E or A.
[0168] [0168] Additional effectors for use in accordance with the invention can be identified by their proximity to the casl genes, for example, although not limited to, within the region 20 kb from the start of the casl gene and 20 kb from the end of the casl gene. gene casl. In certain embodiments, the effector protein comprises at least one HEPN domain and at least 500 amino acids, and wherein the C2c2 effector protein is naturally present in a prokaryotic genome within 20 kb upstream or downstream of a Cas gene or matrix. CRISP Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas66, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2 , Csas5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Ccmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl0, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2 , Csf3, Csf4, homologues thereof or modified versions thereof. In certain exemplary embodiments, the C2c2 effector protein is naturally present in a prokaryotic genome within a
[0169] [0169] In particular embodiments, the Cas enzyme targeting Type VI RNA is C2c2. In other exemplary embodiments, the Cas enzyme targeting Type VI RNA is Cas 13b. In particular embodiments, the homolog or ortholog of a Type VI protein, such as C2c2, as referred to herein, has a sequence homology or identity of at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for example at least 95% with a Type VI protein such as C2c2 (e.g. with based on the wild-type sequence of any of Leptotrichia shahii C2c2, Lachnospiraceae bacterium MAZ2020 c2c2, Lachnospiraceae bacterium NK4A179 Cc2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacterium gallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2, Listeria weihensteensis ( weiphansteensis R9-0317) C2c2, Listeriaceae bacterium (FSL M6-0635) c2c2, Listeria newyorkensis (FSL M6-0635) C2c2, Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2, Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsu latus (DE442) C2c2, Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2). In other embodiments, the homolog or ortholog of a Type VI protein, such as C2c2, as referred to herein, has a sequence identity of at least 30% or at least 40% or at least 50% or at least 60% or at least 60%. at least 70% or at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for example at least 95% with wild type C2c2 (e.g. based on wild type sequence from any of Leptotrichia shahii c2c2, Lachnospiraceae bacterium MA2020 c2c2, Lachnospiraceae bacterium NK4A179 c2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacterium gallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2,
[0170] [0170] In certain other exemplary embodiments, the effector protein of the CRISPR system is a C2c2 nuclease. C2c2 activity may depend on the presence of two HEPN domains. They have been shown to be RNase domains, i.e. nuclease (in particular an endonuclease) snipping RNA. C2c2 HEPN can also target DNA or potentially DNA and/or RNA. Based on the fact that the HEPN domains of C2c2 are at least capable of binding and, in their wild-type form, cutting RNA, it is preferable that the C2c2 effector protein has RNase function. With respect to C2c2 CRISPR systems, reference is made to US Provisional 62/351,662, filed June 17, 2016, and US Provisional 62/376,377, filed August 17, 2016. Reference is also made to Provisional US 62/351,803 , filed on June 17,
[0171] [0171] RNase function in CRISPR systems is known; for example, mRNA targeting has been reported for certain CRISPR-Cas type III systems (Hale et al., 2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139, 945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406417) and offers significant advantages. In the Staphylococcus epidermis type III-A system, transcription between targets results in cleavage of target DNA and its transcripts, mediated by independent active sites in the CaslO0O-Csm ribonucleoprotein effector protein complex (see Samai et al., 2015, Cell, 151, 1164-1174). A CRISPR-Cas system, composition or method targeting RNA through the present effector proteins is thus provided.
[0172] [0172] In one embodiment, the Cas protein may be a C2c2 ortholog of an organism of a genus that includes, but is not limited to, Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,
[0173] [0173] Some methods to identify orthologs of CRISPR-Cas system enzymes may involve identifying tracr sequences in genomes of interest. Identification of tracr sequences can be related to the following steps: Searching for direct repeats or mate tracr sequences in a database to identify a CRISPR region comprising a CRISPR enzyme. Look for homologous sequences in the CRISPR region that flank the CRISPR enzyme in the sense and antisense directions. Look for transcriptional terminators and secondary structures. Identify any sequence that is not a direct repeating sequence or tracermate, but which has more than 50% identity with the direct repeating sequence or tracermate as a potential tracr sequence. Take the potential tracr sequence and analyze the transcriptional terminator sequences associated with it.
[0174] [0174] It will be appreciated that any of the functionalities described herein can be engineered into CRISPR enzymes from other orthologs, including chimeric enzymes comprising fragments from multiple orthologs.
[0175] [0175] In embodiments, the C2c2 protein as referred to herein also encompasses a functional variant of C2c2 or a homolog or ortholog thereof. A "functional variant" of a protein as used herein refers to a variant of that protein that at least partially retains the activity of that protein. Functional variants can include mutants (which can be insertion, deletion or substitution), including polymorphs, etc. Also included in functional variants are fusion products of this protein with another nucleic acid,
[0176] [0176] In one embodiment, the nucleic acid molecule(s) encoding C2c2, or an ortholog or homolog thereof, may be codon-optimized for expression in a eukaryotic cell. A eukaryote may be as discussed herein. Nucleic acid molecules can be modified or unnatural.
[0177] [0177] In one embodiment, the C2c2 or an ortholog or homolog thereof may comprise one or more mutations (and therefore nucleic acid molecules encoding the same may have mutation(s)). Mutations may be artificially introduced mutations and may include, but are not limited to, one or more mutations in a catalytic domain. Examples of catalytic domains with reference to a Cas9 enzyme may include, but are not limited to, RuvC I, RuvC II, RuvC III and HNH domains.
[0178] [0178] In one embodiment, the C2c2, or an ortholog or homolog thereof, may comprise one or more mutations. Mutations may be artificially introduced mutations and may include, but are not limited to, one or more mutations in a catalytic domain. Examples of catalytic domains with reference to a Cas enzyme may include, but are not limited to, HEPN domains.
[0179] [0179] In one embodiment, the C2c2, or an ortholog or homolog thereof, can be used as a generic nucleic acid binding protein fused to or operatively linked to a functional domain. Exemplary functional domains may include, but are not limited to, translational initiator, translational activator, translational repressor, nucleases, in particular ribonucleases, a spliceosome, beads, an inducible/controllable light domain or a chemically inducible/controllable domain.
[0180] [0180] In certain exemplary embodiments, the C2c2 effector protein may be from an organism selected from the group consisting of; Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, and Campylobacter.
[0181] [0181] In certain embodiments, the effector protein may be a Listeria sp. C2c2p, preferably Listeria seeligeria C2c2p, more preferably Listeria seeligeria serovar 1/2b str. SLCC3954 C2c2p and the crRNA sequence can be 44 to 47 nucleotides in length, with a 5' 29-nt forward repeat (DR) and a spacer from 15-nt to l18-nt.
[0182] [0182] In certain embodiments, the effector protein may be a Leptotrichia sp. C2c2p, preferably Leptotrichia shahii C2c2p, more preferably Leptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to 58 nucleotides in length, with a 5' forward repeat of at least 24 nt, such as a 5' forward repeat 24 -28-nt (DR) and a spacer of at least 14 nt, such as a spacer of 14 to 28 nt, or a spacer of at least 18 nt, such as 19, 20, 21, 22, or more nt, such as 18- 28, 19-28, 20-28, 21-28, or 22-28 nt.
[0183] [0183] In certain exemplary embodiments, the effector protein may be a Leptotrichia sPp., Leptotrichia wadei FO0279 or a Listeria sPp., preferably Listeria newyorkensis FSL M6-0635.
[0184] [0184] In certain exemplary embodiments, the C2c2 effector proteins of the invention include, without limitation, the following 21 ortholog species (including various CRISPR loci: Leptotrichia shahii; Leptotrichia wadei (Lw2); Listeria seeligeri; Lachnospiraceae bacterium MA2020; Lachnospiraceae bacterium NK4A179, [Clostridium] aminophilum DSM 10710, Carnobacterium gallinarum DSM 4847, Carnobacterium gallinarum DSM 4847 (according to Loci CRISPR);
[0185] [0185] In certain embodiments, the C2c2 protein according to the invention is or is derived from one of the orthologs, as described in the table below, or is a chimeric protein of two or more of the orthologs, as described in the table below, or is a mutant or variant of one of the orthologs as described in the table below (or a chimeric mutant or variant), including killed C2c2, split C2c2, destabilized C2c2, etc., as defined elsewhere herein, with or without fusion to a domain heterologous/functional.
[0186] [0186] In certain exemplary embodiments, the C2c2 effector protein is selected from Table 1 below.
[0187] [0187] The wild-type protein sequences of the above species are listed in Table 2 below. In certain embodiments, a nucleic acid sequence encoding the C2c2 protein is provided. Table 2 c2c2-2 L. shahii (Lsh) (SEQ. ID No. 1) c2c2-2 L. shahii (Lsh) WP 018451595.1 c2c2-3 L. wadei (Lw2) (SEQ. ID No. 2) c2c2-4 Listeria seeligeri (SEQ. TD No. 3) c2c2-5 1 Lachnospiraceae bacterium MAZ2020 (SEQ. ID No. 4) c2c2-6 2 Lachnospiraceae bacterium NK4A179 c2c2-7 3 Clostridium aminophilum DSM 10710 (SEQ. I.D.
[0188] [0188] In one embodiment of the invention, an effector protein is provided that comprises an amino acid sequence with at least 80% sequence homology to the wild-type sequence of any of the bacteria Leptotrichia shahii C2c2, Lachnospiraceae MA2020 C2c2, Lachnospiraceae bacterium NK4A179 C2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacterium gallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2, Listeria weihenstephanensis (FSL R9-0317) c2c2, Listeriaceae bacterium (FSL M6-0635) C2c2, Listeria FSL Mork- 0635), Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2, Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2, Leptotrichia wadei (Lw2) C2c2 or Listeria seeliger.
[0189] [0189] In one embodiment of the invention, the effector protein comprises an amino acid sequence having at least 80% sequence homology to a Type VI effector protein consensus sequence, including, but not limited to, a consensus sequence herein described.
[0190] [0190] In accordance with the invention, a consensus sequence can be generated from multiple C2c2 orthologs, which can assist in locating conserved amino acid residues and motifs, including, but not limited to, catalytic residues and HEPN motifs. in C2c2 orthologs that mediate C2c2 function. One such consensus sequence, generated from the 33 orthologs mentioned above, using the Geneious alignment is: MKISKVXXXVXKKXXXGKLXKXVNERNRXAKRLSNXLBKY IXXIDKIXKKEXXKKFXAX EEITLKLNOQXXXBXLXKAXXDLRKDNXYSXJKKILHNEDINXEEXELLINDXLEKLXKI ESXKYSYQKXXXNYXMSVOQEHSKKSIXRIXESAKRNKEALDKFLKEYAXLDPRMEXLAK LRKLLELYFYFKNDXIXXEEEXNVXXHKXLKENHPDFVEXXXNKENAELNXYAIEXKKJ LKYYFPXKXAKNSNDKIFEKQELKKXWIHQJENAVERILLXXGKVXYKLQOXGYLAELWK IRINEIFIKYIXVGKAVAXFALRNXXKBENDILGGKIXKKLNGITSFXYEKIKAEEILO REXAVEVAFAANXLYAXDLXXIRXSILQFFGGASNWDXFLFFHFATSXISDKKWNAELI XXKKIGLVIREKLYSNNVAMFYSKDDLEKLLNXLXXFXLRASQVPSFKKVYVRXBFPQON LLKKFNDEKDDEAYSAXYYLLKEIYYNXFLPYFSANNXFFFXVKNLVLKANKDKEFXXAF XDIREMNXGSPIEYLXXTQOXNXXNEGRKKEEKEXDF IKFLLQIFXKGFDDYLKNNXXFI LKFIPEPTEXIEIXXELQAWY IVGKFLNARKXNLLGXFXSYLKLLDDIELRALRNENIK YQOSSNXEKEVLEXCLELIGLLSLDLNDYFBDEXDFAXYJGKXLDFEKKXMKDLAELXPY DONDGENPIVNRNIXLAKKYGTLNLLEKIXDKVSEKEIKEYYELKKEIEEYXXKGEELH EEWXOXKNRVEXRDILEYXEELXGQIINYNXLXNKVLLYFQOLGLHYLLLDILGRLVGYT GIWERDAXLYQIAAMYXNGLPEYIXXKKNDKYKDGQIVGXKINXFKXDKKXLYNAGLEL FENXNEHKNIXIRNYIAHENYLSKAESSLLXYSENLRXLFSYDRKLKNAVXKSLINILL
[0191] [0191] In another non-limiting example, a sequence alignment tool to assist in generating a consensus sequence and identifying conserved residues is the MUSCLE alignment tool (www.ebi.ac.uk/Tools/msa/muscle/ ). For example, using MUSCLE, the following conserved amino acid sites among C2c2 orthologs can be identified in Leptotrichia wadei C2c2: K2; K5; V6; E301; L331; 1335; N341; G351; K352; E375; L392; L396; D403; F446; I466; 1470; R474 (HEPN); H475; H479 (HEPN), E508; P556; L561; 1595; Y596; F600; Y669; I673; F681; L685; Y761; L676; L779; Y782; L836; D847; Y863; L869; 1872; K879; 1933; L954; I958; R961; Y965; E970; R971; D972; R1046 (HEPN), H1051 (HEPN), Y1075; D1076; K1078; K1080; 11083; 11090.
[0192] [0192] An example sequence alignment of HEPN domains showing highly conserved residues is shown in FIG. 50.
[0193] [0193] In certain example embodiments, the RNA-targeting effector protein is a Type VI-B effector protein, such as Casl3b and Group 29 or Group 30 proteins. In certain example embodiments, the RNA targeting effector protein comprises one or more HEPN domains.
[0194] [0194] In certain example embodiments, the wild-type sequence of the Casl3b ortholog is found in Table 4a or 4b below.
[0195] [0195] In certain example embodiments, the RNA-targeted effector protein is a Casl3c effector protein, as disclosed in Provisional Patent Application US 62/525,165 filed June 26, 2017 and PCT Application US 2017/047193 filed 16 August 2017. Sequences from wild-type orthologs of Casl3c are provided in Table 5 below.
[0196] [0196] In certain example embodiments, the assays may comprise multiple Casl2 orthologs or one or more orthologs in combination with one or more Cas1l3 orthologs. In certain example embodiments, the Casl2 orthologs are Cpfl orthologs. In certain other example embodiments, the Casl2 orthologs are C2cl orthologs.
[0197] [0197] The present invention encompasses the use of a Cpfl effector protein, derived from a Cpfl locus indicated as a V-A subtype. Here, these effector proteins are also called "Cpflp", for example a Cpfl protein (and this effector protein or Cpfl protein or protein derived from a Cpfl locus is also called a "CRISPR enzyme"). Currently, the V-A loci subtype encompasses casl, cas2, a distinct gene denoted cpfl, and a CRISPR array. Cpfl (Cpfl protein associated with CRISPR, subtype PREFRAN) is a large protein (about 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9, along with an equivalent to the characteristic arginine-rich cluster of Cas9 . However, Cpfl lacks the HNH nuclease domain that is present in all Cas9 proteins and the RuvC-like domain is contiguous in the Cpfl sequence, in contrast to Cas9, where it contains long inserts, including the HNH domain. Therefore, in particular embodiments, the CRISPR-Cas enzyme comprises only a RuvC-like nuclease domain.
[0198] [0198] The programmability, specificity, and collateral activity of RNA-guided Cpfl also make it an ideal switchable nuclease for nonspecific cleavage of nucleic acids. In one embodiment, a Cpfl system is designed to provide for and take advantage of non-specific RNA collateral cleavage. In another embodiment, a Cpfl system is designed to provide and take advantage of SSDNA's non-specific collateral cleavage. Therefore, engineered Cpfl systems provide platforms for nucleic acid detection and transcriptome manipulation. Cpfl was developed to be used as a mammalian transcriptional binding and knockdown tool. Cpfl is capable of robust collateral cleavage of RNA and ssDNA when activated by binding to sequence-specific targeted DNA.
[0199] [0199] The terms "ortholog" (also referred to herein as "ortholog") and "homologous" (also referred to herein as "homologous") are well known in the art. By way of additional guidelines, a "homolog" of a protein as used herein is a protein of the same species that performs the same or similar function as the protein of which it is a homolog. Homologous proteins can, but need not, be structurally related, or are only partially structurally related. An "ortholog" of a protein as used herein is a protein of a different species that performs the same or a similar function as the protein of which it is an ortholog. Orthologous proteins can, but need not, be structurally related, or are only partially structurally related. Homologs and orthologs can be identified by homology modeling (see, for example, Greer, Science vol. 228 (1985 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "structural BLAST" Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a "structural BLAST": using structural relationships to infer function. Protein Sci. 2013 Apr;22(4):359-66. doi: 10.1002/pro.2225 See also Shmakov et (2015) for application in the field of CRISPR-Cas loci Homologous proteins can, but need not, be structurally related, or are only partially structurally related.
[0200] [0200] The Cpfl gene is found in several diverse bacterial genomes, typically at the same location with the casl, cas2 and cas4 genes and a CRISPR cassette (eg FNFX1 1431-FNFX1 1428 from Francisella cf. Novicida Fxl). Thus, the layout of this new putative CRISPR-Cas system appears to be similar to type II-B. In addition, similar to Cas9, the Cpfl protein contains a “readily identifiable C-terminal region that is homologous to the ORF-B transposon and includes an active RuvC-like nuclease, an arginine-rich region, and a Zn finger (absent in Cas9). .
[0201] [0201] In particular embodiments, the effector protein is a Cpfl effector protein from an organism of a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium , Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus,
[0202] [0202] In other particular embodiments, the Cpfl effector protein is from a selected S&S organism. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S&S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; ss. auricularis, Ss. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii.
[0203] [0203] The effector protein may comprise a chimeric effector protein comprising a first fragment of a first effector protein ortholog (e.g. a Cpfl) and a second fragment of a second effector protein ortholog (e.g. a Cpfl) and in that the first and second effector protein orthologs are different. At least one of the first and second orthologs of effector proteins (e.g. a Cpfl1) may comprise an effector protein (e.g. a Cpfl) from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter , Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella,
[0204] [0204] In a more preferred embodiment, the Cpflp is derived from a selected bacterial species from Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC244 17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MAZ020, Candidatus Methanoplasma termitum, Eubacterium elegens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae. In certain embodiments, the Cpflp is derived from a bacterial species selected from
[0205] [0205] In some embodiments, the Cpflp is derived from an organism of the genus Eubacterium. In some embodiments, the CRISPR effector protein is a Cpfl protein derived from an organism of the Eubacterium rectale bacterial species. In some embodiments, the amino acid sequence of the cpf1 effector protein corresponds to NCBI Reference Sequence WP 055225123.1, NCBI Reference Sequence WP 055237260.1, NCBI Reference Sequence WP 055272206.1 or GenBank ID OLA16049.1. In some embodiments, the Cpfl effector protein has a sequence homology or sequence identity of at least 60%, more particularly at least 70, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, as for example at least 95% with the NCBI reference sequence WP 055225123.1, the NCBI reference sequence WP 055237260.1, the NCBI reference sequence WP 055272206.1 or CGenBank ID OLA1I6049.1. The skilled person will understand that this includes truncated forms of the Cpfl protein, whereby sequence identity is determined along the length of the truncated form. In some embodiments, the Cpfl effector recognizes the PAM sequence of TTTN or CTTN.
[0206] [0206] In particular embodiments, the Cpfl homolog or ortholog as referred to herein has a homology or sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, as for example at least 95% with Cpfl. In other embodiments, the Cpfl homolog or ortholog as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for example at least 95% with the wild-type Cpfl. When the Cpfl has one or more mutations (mutated), the homolog or ortholog of said Cpfl, as referred to herein, has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90% , such as at least 95% with the mutated Cpfl.
[0207] [0207] In one embodiment, the Cpfl protein may be an ortholog of an organism of a genus that includes, but is not limited to, Acidaminococcus sp, Lachnospiraceae bacterium, or Moraxella bovoculi; in particular embodiments, the Cas type V protein may be an ortholog of an organism of a species that includes, but is not limited to, Acidaminococcus sp. BV3L6; Bacteria Lachnospiraceae ND2006 (LbCpf1) or Moraxella bovoculi
[0208] [0208] In particular embodiments, the Cpfl protein of the invention has a homology or sequence identity of at least 60%, more particularly at least 70, such as at least 80%, more preferably at least 85%, even more preferably at least 90 %, for example at least 95% with FnCpfl, AsCpfl or LbCpfl. In other embodiments, the Cpfl protein as referred to herein has a sequence identity of at least 60%, such as at least 70%, more particularly at least 80%, more preferably at least 85%, even more preferably at least 90 %, e.g. at least 95% with wild type ASsCpfl or LbCpfl. In particular embodiments, the Cpfl protein of the present invention has less than 60% sequence identity to FnCpfl. The skilled person will understand that this includes truncated forms of the Cpfl protein, whereby sequence identity is determined along the length of the truncated form.
[0209] [0209] The present invention encompasses the use of C2cl effector proteins, derived from a C2cl locus indicated as subtype V-B. Here, these effector proteins are also called "C2clp", for example a C2cl protein (and this effector protein or C2cl protein or protein derived from a C2cl locus is also called a "CRISPR enzyme"). Currently, the V-B loci subtype encompasses a casl-Cas4, cas2 fusion, a distinct gene denoted C2cl, and a CRISPR arrangement. C2cl (C2cl protein associated with CRISPR) is a large protein (about 1100 - 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9, along with an equivalent to the characteristic arginine-rich cluster of Cas9. However, C2cl lacks the HNH nuclease domain that is present in all Cas9 proteins and the RuvC-like domain is contiguous in the C2cl sequence, in contrast to Cas9, where it contains long inserts, including the HNH domain. Therefore, in particular embodiments, the CRISPR-Cas enzyme comprises only a RuvC-like nuclease domain.
[0210] [0210] C2cl proteins (also known as Casl12b) are RNA-guided nucleases. Its cleavage relies on a tracr RNA to recruit a chi RNA comprising a leader sequence and a forward repeat, where the leader sequence hybridizes to the target nucleotide sequence to form a DNA/RNA heteroduplex. Based on current studies, C2cl nuclease activity also requires PAM sequence recognition. C2cl PAM sequences are T-rich sequences. In some embodiments, the PAM sequence is 5' TTN 3' or 5' ATTN 3', where N is any nucleotide. In a particular embodiment, the PAM sequence is 5' TTC 3'. In a particular embodiment, the PAM is in the sequence of Plasmodium falciparum.
[0211] [0211] C2cl creates a staggered cut at the target location, with a 5' overhang or a “sticky end” on the distal side of the PAM of the target sequence. In some embodiments, the 5' overhang is 7 nt. See Lewis and Ke, Mol Cell. 2017 Feb 2;65(3):377-379.
[0212] [0212] The invention provides C2cl (Type V-B; Casl2b) effector proteins and orthologs. The terms "ortholog" (also referred to herein as "ortholog") and "homologous" (also referred to herein as "homologous") are well known in the art. By way of additional guidelines, a "homolog" of a protein as used herein is a protein of the same species that performs the same or similar function as the protein of which it is a homolog. Homologous proteins can, but need not, be structurally related, or are only partially structurally related. An "ortholog" of a protein as used herein is a protein of a different species that performs the same or a similar function as the protein of which it is an ortholog. Orthologous proteins can, but need not, be structurally related, or are only partially structurally related. Homologs and orthologs can be identified by homology modeling (see, for example, Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "structural BLAST" Dey F , Cliff Zhang Q, Petrey D, Honig B. Toward a "structural BLAST": using structural relationships to infer function. Protein Sci. 2013 Apr;22(4):359-66. doi: 10.1002/pro.2225 See also Shmakov et al. (2015) for application in the field of CRISPR-Cas loci. Homologous proteins can, but need not, be structurally related, or are only partially structurally related.
[0213] [0213] The C2cl gene is found in several diverse bacterial genomes, typically at the same location with the casl, cas2 and cas4 genes and a CRISPR cassette. Thus, the layout of this new putative CRISPR-Cas system appears to be similar to type II-B. In addition, similar to Cas9, the C2cl protein contains an active RuvC-type nuclease, an arginine-rich region and a Zn finger (absent in Casº9).
[0214] [0214] In particular embodiments, the effector protein is a C2cl effector protein from an organism of a genus comprising Alicyclobacillus, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Candidatus, Desulfatirhabdium, Citrobacter, Elusimicrobia, Methylobacterium, Omnitrophica, Phycisphaerae, Planctomycetes, Spirochaetes, and Verrucomicrobiaceae.
[0215] [0215] In other particular embodiments, the C2cl effector protein is from a selected species of Alicyclobacillus acidoterrestris (eg ATCC 49025), Alicyclobacillus contaminans (eg DSM 17975), Alicyclobacillus macrosporangiidus (eg DSM 17980), Bacillus hisashii Cc4 strain, Candidatus Lindowbacteria bacterium RIFCSPLOWO2, Desulfovibrio inopinatus (eg DSM 10711), Desulfonatronum thiodismutans (eg MLF-l1 strain), E&Elusimicrobia bacterium RIFOXYALl2, Omnitrophica WOR 2 bacterium RIFCSPHIGHO2, Opitutaceae bacterium TAV5, Phycisphaerae bacterium ST-NAGAB-bacterium D1, Planctomycetes bacterium
[0216] [0216] The effector protein may comprise a chimeric effector protein comprising a first fragment of a first effector protein ortholog (e.g. a C2cl) and a second fragment of a second effector protein ortholog (e.g. a C2cl) and in that the first and second effector protein orthologs are different. At least one of the first and second orthologs of effector proteins (e.g. a C2cl) may comprise an effector protein (e.g. a C2cl) from an organism comprising Alicyclobacillus, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Candidatus, Desulfatirhabdium, Elusimicrobia, Citrobacter, Methylobacterium, Omnitrophicai, Phycisphaerae, Planctomycetes, Spirochaetes, and Verrucomicrobiaceae; for example, a chimeric effector protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a C2cl from an organism comprising Alicyclobacillus, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Candidatus, Desulfatirhabdium, Elusimicrobia, Citrobacter, Methylobacterium, Omnitrophicai, Phycisphaerae, Planctomycetes, Spirochaetes, and Verrucomicrobiaceae in which the first and second fragments are not from the same bacterium; for example a chimeric effector protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a C2cl of Alicyclobacillus acidoterrestris (e.g. ATCC 49025), Alicyclobacillus contaminans (e.g. DSM 17975) , Alicyclobacillus macrosporangiidus (e.g. DSM 17980), Bacillus hisashii strain Cc4, Candidatus Lindowbacteria bacterium RIFCSPLOWO 2, Desulfovibrio inopinatus (e.g. DSM 10711), Desulfonatronum thiodismutans (e.g. MLF-11 strain), Elusimicrobia bacterium RIFOXYAl2, Omnitrophica WOR 2 bacterium RIFCSPHIGHO2, Opitutaceae bacterium TAV5, Phycisphaerae bacterium ST-NAGAB-D1, Planctomycetes bacterium RBG 13 46 10, Spirochaetes bacterium GWB1 27 13, Verrucomicrobiaceae bacterium UBA2429, Tuberibacillus calidus (e.g. DSM 17572), Bacillus thermoamylovorans (e.g. strain B4166), Brevibacillus sp. CF1112, Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (eg DSM 18734), Alicyclobacillus herbarius (eg DSM 13609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500), Methylobacterium nodulans (eg , ORS 2060), in which the first and second fragments are not from the same bacterium.
[0217] [0217] In a more preferred embodiment, the C2clp is derived from a selected bacterial species of Alicyclobacillus acidoterrestris (e.g. ATCC 49025), Alicyclobacillus contaminans (e.g. DSM 17975), Alicyclobacillus macrosporangiidus (e.g. DSM 17980), Bacillus hisashii Cc4 strain, Candidatus Lindowbacteria bacterium RIFCSPLOWO2, Desulfovibrio inopinatus (eg DSM 10711), Desulfonatronum thiodismutans (eg MLF-l1 strain), Elusimicrobia bacterium RIFOXYA1l2, Omnitrophica WOR 2 bacterium RIFCSPHIGHO2, Opitutaceae bacterium TAV5, Phycisphaerae bacterium ST-NAGAB-bacterium D1, Planctomycetes bacterium RBG 13 46 10, Spirochaetes bacterium GWB1 27 13, Verrucomicrobiaceae bacterium UBAZ429, Tuberibacillus calidus (e.g. DSM 17572), Bacillus thermoamylovorans (e.g. strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (eg DSM 18734), Alicyclobacillus herbarius (eg DSM 13609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500), Methylobacterium nodulans (eg , ORS 2060). In certain embodiments, the C2clp is derived from a selected bacterial species of Alicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacillus contaminans (e.g., DSM 17975).
[0218] [0218] In particular embodiments, the C2cl homolog or ortholog as referred to herein has a homology or sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, as for example at least 95% with C2cl. In other embodiments, the C2cl homolog or ortholog as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for example at least 95% with the wild-type C2cl. When the C2cl has one or more mutations (mutated), the homolog or ortholog of said C2cl, as referred to herein, has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90% , for example at least 95% with the mutated C2cl.
[0219] [0219] In one embodiment, the C2cl protein may be an ortholog of an organism of a genus that includes, but is not limited to, Alicyclobacillus, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Candidatus, Desulfatirhabdium, Elusimicrobia, Citrobacter, Methylobacterium, Omnitrophicai, Phycisphaerae, Planctomycetes, Spirochaetes, and Verrucomicrobiaceae; in particular embodiments, the Cas type V protein may be an ortholog of an organism of a species that includes, but is not limited to, Alicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacillus contaminans (e.g., DSM 17975), Alicyclobacillus macrosporangiidus (e.g., e.g. DSM 17980), Bacillus hisashii strain Cc4, Candidatus Lindowbacteria bacterium RIFCSPLOWO2, Desulfovibrio inopinatus (e.g. DSM 10711), Desulfonatronum thiodismutans (e.g. MLF-l1 strain), Elusimicrobia bacterium RIFOXYAl2, Omnitrophica WOR 2 bacterium RIFCSPHIGHO2, Opitutaceae bacterium TAVS5, Phycisphaerae bacterium ST-NAGAB-D1, Planctomycetes bacterium RBG 13 46 10, Spirochaetes bacterium GWB1 27 13, Verrucomicrobiaceae bacterium UBAZ429, Tuberibacillus calidus (eg, DSM 17572), Bacillus thermoamylovorans (e.g. strain B4166), Brevibacillus sp. CFl112, Bacillus sp.
[0220] [0220] In particular embodiments, the C2cl protein of the invention has a homology or sequence identity of at least 60%, more particularly at least 70, such as at least 80%, more preferably at least 85%, even more preferably at least 90 %, eg at least 95% with AacC2cl or Bthc2cl. In other embodiments, the C2cl protein, as referred to herein, has a sequence identity of at least
[0221] [0221] The programmability, specificity and collateral activity of RNA-guided C2cl also make it an ideal switchable nuclease for nonspecific cleavage of nucleic acids. In one embodiment, a C2cl system is designed to provide for and take advantage of non-specific RNA collateral cleavage. In another embodiment, a C2cl system is designed to provide and take advantage of SSDNA's non-specific collateral cleavage. Therefore, engineered C2cl systems provide platforms for nucleic acid detection and transcriptome manipulation and induce cell death. C2cl was developed to be used as a mammalian transcriptional binding and knockdown tool. C2cl is capable of robust collateral cleavage of RNA and ssDNA when activated by binding to sequence-specific targeted DNA.
[0222] [0222] In one embodiment, the C2c1l system is engineered to cleave RNA non-specifically in a subset of cells distinguishable by the presence of an aberrant DNA sequence, for example, where cleavage of aberrant DNA may be incomplete or ineffective. In a non-limiting example, a DNA translocation present in a cancer cell that promotes cellular transformation is targeted. While a subpopulation of cells that undergoes chromosomal DNA and repair may survive, nonspecific ribonuclease collateral activity advantageously leads to cell death of potential survivors.
[0223] [0223] Collateral activity has recently been leveraged into a highly sensitive and specific nucleic acid detection platform called SHERLOCK, useful for many clinical diagnoses (Gootenberg, JS et al. Nucleic acid detection with CRISPR-Casl3a/C2c2. Science 356, 438 -442 (2017)).
[0224] [0224] In accordance with the invention, engineered C2cl systems are optimized for DNA or RNA endonuclease activity and can be expressed in mammalian cells and targeted to effectively knock down reporter molecules or transcripts in cells. GUIDE SEQUENCES
[0225] [0225] As used in this document, the term "guide sequence", "crRNA", "guide RNA" or "single guide RNA"
[0226] [0226] In some embodiments, a nucleic acid targeting guide is selected to reduce the degree of secondary structure within the nucleic acid targeting guide. In some embodiments, approximately or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less of the nucleic acid targeting guide nucleotides participate in self-complementary base pairing when ideally folded. optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of such an algorithm is mFoldy, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the RNAfold online server, developed at the Institute of Theoretical Chemistry of the University of Vienna, using the centroid structure prediction algorithm (see, for example, AR Gruber et al., 2008, Cell 106( 1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62 ).
[0227] [0227] In certain embodiments, a lead RNA or crRNA may comprise, essentially consist of, or consist of a direct repeat (DR) sequence and a lead sequence or spacer sequence. In certain embodiments, the leader RNA or oO CrRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a leader sequence or spacer sequence. In certain embodiments, the forward repeat sequence may be located upstream (i.e., 5') of the leader sequence or spacer sequence. In other embodiments, the forward repeat sequence may be located downstream (i.e., 3') of the guide sequence or spacer sequence.
[0228] [0228] In certain embodiments, the CcrRNA comprises a stem loop, preferably a single stem loop. In certain embodiments, the direct repeat sequence forms a stem loop, preferably a single stem loop.
[0229] [0229] In certain embodiments, the guide RNA spacer length is 15 to 35 nt. In certain embodiments, the length of the guide RNA spacer is at least 15 nucleotides. In certain embodiments, the spacer length is 15 to 17 nt, e.g. 15, 16 or 17 nt, 17 to 20 nt, e.g. 17, 18, 19 or 20 nt, 20 to 24 nt, e.g. e.g. 20, 21, 22, 23 or 24 nt, from 23 to 25 nt, e.g. 23, 24 or 25 nt, from 24 to 27 nt, e.g. 24, 25, 26 or 27 nt, from 27 to 30 nt, for example 27, 28, 29 or 30 nt, from 30 to 35 nt, for example 30, 31, 32, 33, 34 or 35 nt, or 35 nt or more.
[0230] [0230] In general, the CRISPR-Cas system, CRISPR-Cas9 or CRISPR system can be used in earlier documents such as WO 2014/093622 (PCT/US2013/074667) and collectively refers to transcripts and other elements involved in expression or targeting the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, in particular a Cas9 gene in the case of CRISPR-Cas9, a tracr sequence (trans-activating CRISPR) (e.g. tracrRNA or a active partial tracrRNA), a tracr-mate sequence (which includes a "direct repeat"
[0231] [0231] In the embodiments of the invention, the terms guide sequence and guide RNA, that is, RNA capable of guiding Cas to a target genomic locus, are used interchangeably as in the documents cited above, such as WO 2014/093622 (PCT/US2013 /074667). In general, a chia sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when ideally aligned using a suitable alignment algorithm, is about 50%, 60%
[0232] [0232] In some embodiments of CRISPR-Cas systems, the degree of complementarity between a guide sequence and its corresponding target sequence can be around 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or 100%; a guide or RNA or SgRNA can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length; or guide or RNA or SsgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length; and advantageously the tracr RNA is 30 or 50 nucleotides in length. However, one aspect of the invention is to reduce off-target interactions, for example,
[0233] [0233] In certain embodiments, guides to the invention comprise non-naturally occurring nucleic acids and/or non-naturally occurring nucleotides and/or nucleotide analogs and/or chemical modifications. non-naturally occurring nucleic acids may include, for example, mixtures of naturally occurring and non-naturally occurring nucleotides.
[0234] [0234] In certain embodiments, the CRISPR system as provided herein may make use of a crRNA or polynucleotide analog comprising a guide sequence, wherein the polynucleotide is an RNA, a DNA, or a mixture of RNA and DNA and/or wherein the polynucleotide comprises one or more nucleotide analogues. The sequence may comprise any structure, including, without limitation, a structure of a native crRNA, such as a bulge, hairpin, or rod loop structure. In certain embodiments, the polynucleotide comprising the leader sequence forms a duplex with a second polynucleotide sequence which may be an RNA or DNA sequence.
[0235] [0235] In certain embodiments, use is made of chemically modified guide RNAs. Examples of chemical modifications of guide RNA include, without limitation, incorporation of 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP) into one or more terminal nucleotides. Such chemically modified lead RNAs may comprise greater stability and greater activity compared to unmodified chias RNAs, although on-target versus off-target specificity is not predictable. (See Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, published online June 29, 2015). Chemically modified guide RNAs further include, without limitation, RNAs with phosphorothioate bonds and blocked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring.
[0236] [0236] In some embodiments, a guide sequence is approximately or more than 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In some embodiments, a leader sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. Preferably, the leader sequence is 10 to 30 nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including the leader sequence to be tested, can be provided to a host cell with the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, as in the Surveyor assay. Likewise, the cleavage of a target RNA can be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the chia sequence to be tested and a control guide sequence different from the test guide and comparing the binding or rate of cleavage in the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art.
[0237] [0237] In some embodiments, the guide modification is a chemical modification, an insertion, a deletion, or a split. In some embodiments, the chemical modification includes, but is not limited to, incorporation of 2'-O-methyl (M) analogs, 2'-deoxy analogs, 2-
[0238] [0238] In some embodiments, the handle of the 5' handle of the guide is modified. In some embodiments, the loop of the 5' loop of the guide is modified to have a deletion, an insertion, a split, or chemical modifications. In certain embodiments, the loop comprises 3, 4 or 5 nucleotides. In certain embodiments, the loop comprises the sequence of UCUU, UUUU, UAUU, or UGUU.
[0239] [0239] A guide sequence and therefore a nucleic acid targeting guide RNA can be selected to target any target nucleic acid sequence. In the context of forming a CRISPR complex, "target sequence" refers to a sequence in which a leader sequence is designed to have complementarity, where hybridization between a target sequence and a leader sequence promotes formation of a CRISPR complex. . A target sequence may comprise RNA polynucleotides. The term "target RNA" refers to an RNA polynucleotide that is or comprises the target sequence. In other words, the target RNA may be an RNA polynucleotide or a part of an RNA polynucleotide in which a part of the gRNA, i.e., the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising the CRISPR effector protein and a gRNA must be targeted. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. The target sequence can be DNA. The target sequence can be any RNA sequence. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA ( MiRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double-stranded RNA (dAsRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRKNA), and small cytoplasmic RNA (SCRNA). In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA and I1IncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a P-pre-mRNA molecule.
[0240] [0240] In certain embodiments, the spacer length of the guide RNA is less than 28 nucleotides. In certain embodiments, the guide RNA spacer length is at least 18 nucleotides and less than 28 nucleotides. In certain embodiments, the spacer length of the guide RNA is between 19 and 28 nucleotides. In certain embodiments, the length of the guide RNA spacer is between 19 and 25 nucleotides. In certain embodiments, the length of the guide RNA spacer is 20 nucleotides. In certain embodiments, the length of the guide RNA spacer is 23 nucleotides. In certain embodiments, the length of the guide RNA spacer is 25 nucleotides.
[0241] [0241] In certain embodiments, modulations of cleavage efficiency can be exploited by introducing mismatches, e.g. 1 or more mismatches, such as 1 or 2 mismatches between the spacer sequence and the target sequence, including the position of the mismatch along the spacer/target. The more central (ie not 3' or 5'), for example, a double mismatch, the more the cleavage efficiency is affected. Therefore, by choosing the mismatch position along the spacer, the cleavage efficiency can be modulated. By way of example, if less than 100% cleavage of targets is desired (e.g. in a population of cells), 1 or more, preferably 2, mismatches between spacer and target sequence can be introduced into the spacer sequences. The more central along the spacer of the mismatch position, the lower the percentage of cleavage.
[0242] [0242] In certain exemplary embodiments, cleavage efficiency can be exploited to design unique guides that can distinguish two or more targets that vary by a single nucleotide, such as a single nucleotide polymorphism (SNP), variation, or (point) mutation. The CRISPR effector may have reduced sensitivity to SNPs (or other single nucleotide variations) and continue to cleave SNP targets with a certain level of efficiency. Thus, for two targets, or a set of targets, a guide RNA can be designed with a nucleotide sequence that is complementary to one of the targets, i.e. the SNP on the target. Guide RNA is even designed to have a synthetic mismatch. As used herein, a "synthetic mismatch" refers to an unnatural mismatch that is introduced upstream or downstream of the naturally occurring SNP, such as a maximum of 5 nucleotides upstream or downstream, e.g. 4, 3, 2 or 1 nucleotide upstream or downstream, preferably at most 3 nucleotides upstream or downstream, more preferably at most 2 nucleotides upstream or downstream, most preferably 1 nucleotide upstream or downstream (i.e. adjacent to the SNP) . When the CRISPR effector binds to the SNP on the target, only a single mismatch will be formed with the synthetic mismatch and the CRISPR effector will continue to be activated and a detectable signal will be produced. When the guide RNA hybridizes to an off-target SNP, two mismatches are formed, the SNP mismatch and the synthetic mismatch and no detectable signal is generated. Thus, the systems disclosed herein can be designed to distinguish SNPs within a population. For example, the systems can be used to distinguish pathogenic strains that differ by a single SNP or detect certain disease-specific SNPs, such as, without limitation, disease-associated SNPs, such as, without limitation, cancer-associated SNPs.
[0243] [0243] In certain embodiments, RNA chi is designed so that the SNP is located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of the spacer sequence (starting at the 5' end). In certain embodiments, the guide RNA is designed so that the SNP is located at positions 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the spacer sequence (starting at the 5' end). In certain embodiments, the guide RNA is designed so that the SNP is located at positions 2, 3, 4, 5, 6, or 7 of the spacer sequence (starting at the 5' end). In certain embodiments, the guide RNA is designed so that the
[0244] [0244] In certain embodiments, the chi RNA is designed so that the mismatch (e.g., synthetic mismatch, i.e., an additional mutation in addition to an SNP) is located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 of spacer sequence (starting at the 5' end). In certain embodiments, the guide RNA is designed so that the mismatch is located at positions 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the spacer sequence (starting at the 5' end). In certain embodiments, the guide RNA is designed so that the mismatch is located at positions 4, 5, 6, or 7 of the spacer sequence (starting at the 5' end). In certain embodiments, the guide RNA is designed so that the mismatch is located at position 5 of the spacer sequence (starting at the 5' end).
[0245] [0245] In certain embodiments, the guide RNA is designed so that the mismatch is located 2 nucleotides upstream of the SNP (ie, one intervening nucleotide).
[0246] [0246] In certain embodiments, the guide RNA is designed so that the mismatch is located 2 nucleotides downstream of the SNP (ie, one intervening nucleotide).
[0247] [0247] In certain embodiments, the chi RNA is designed such that the mismatch is located at position 5 of the spacer sequence (starting at the 5' end) and the SNP is located at position 3 of the spacer sequence (starting at the 5' end) .
[0248] [0248] The modalities described herein comprise inducing one or more nucleotide modifications in a eukaryotic cell (in vitro, i.e., in an isolated eukaryotic cell) as discussed herein comprising delivering to the cell a vector as discussed herein. The mutation(s) may (m) include the inclusion, deletion or substitution of one or more nucleotides in each cell target sequence(s) via the guide RNA(s). Mutations may include the introduction, deletion or substitution of 1-75 nucleotides in each target sequence of said cell(s) through the guide RNA(s). Mutations may include the introduction, deletion or replacement of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides in each target sequence of said cell(s) through the guide RNA(s). Mutations may include the introduction, deletion or substitution of 5 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides in each target sequence of said cell(s) via the guide RNA(s) Mutations include the introduction, deletion or substitution of 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides in each target sequence of said cell(s) via guide RNA(s) ). Mutations can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides in each target sequence of the( s) referred cell(s) through the guide RNA(s). Mutations may include the introduction, deletion or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each target sequence of said cell(s) through the guide RNA(s).
[0249] [0249] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed to one or more Cas proteins) results in cleavage at or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of the) target sequence, but may depend on, for example,
[0250] [0250] In certain exemplary embodiments, the signal amplification CRISPR effector protein is an effector protein of the CRISPR-Cas Type III FA system. In certain exemplary embodiments, the CRISPR-Cas Type III-A effector protein is Csm6. Csm6 works with the Csm multiprotein effector complex, but is not part of the complex (see, for example, US20170198286 Al; WO2016035044Al; M. Kazlauskiene et al., Science 10.1126/science.aao0100 (2017); and Niewoehner et al. 2017 , bioRxiv preprint first published online June 23, 2017; doi: dx.doi.org/10.1101/153262).
[0251] [0251] In Staphylococcus epidermidis, the Csm complex (SeCsm) is composed of Casl10, Csm2, Csm3, Csm4 and Csm5 proteins. The CRISPR-Cas type III-A system has been shown to have RNA cleavage activity in vitro and in the cell using the Csm complex for Streptococcus thermophilus (St) (see, for example, US20170198286 A1).
[0252] [0252] Type III-A CRISPR-Cas systems include Streptococcus thermophilus (GenBank KM222358), DGCC7710 (GenBank AWVZO01000003), LMD-9 (GenBank NCO008532), Staphylococcus epidermidis RP62a (GenBank NCO002976), Enterococcus italicus DSM15952 (GenBank
[0253] [0253] Csm6 has been shown to be an SSRNA-specific endoribonuclease and the structural basis for this activity has been determined (Niewoehner and Jinek, 2016, Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. RNA 22:318 -329).
[0254] [0254] In some embodiments, one or more elements of a nucleic acid targeting system of the present invention is derived from a particular organism that comprises an endogenous CRISPR RNA targeting system.
[0255] [0255] In certain exemplary embodiments, the CRISPR-based detection systems described herein comprise a Csm6 protein comprising at least one HEPN domain, including, but not limited to the HEPN domains described herein, HEPN domains known in the art (Niewoehner and Jinek , 2016) and domains recognized as HEPN domains by comparison with consensus sequence motifs. Several such domains are provided here. In a non-limiting example, a consensus sequence can be derived from the C2c2 or Casl3b ortholog sequences provided herein. In certain exemplary embodiments, the Csm6 protein comprises a single HEPN domain. In certain other exemplary embodiments, the Csm6 protein comprises two HEPN domains.
[0256] [0256] In an exemplary embodiment, the Csm6 protein comprises one or more HEPN domains comprising an RxxxxH motif sequence. The RxxxxH motif sequence can be, without limitation, from a HEPN domain described herein or a HEPN domain known in the art. The RxxxxH motif sequences further include motif sequences created by combining portions of two or more HEPN domains. As noted, consensus sequences can be derived from the sequences of the orthologs disclosed herein. In certain embodiments, the HEPN domain comprises a conserved R-X4-6-H motif ( Anantharaman et al., Biol Direct. 2013 Jun 15; 8:15; and Kim et al., Proteins. 2013 Feb; 81(2) :261-70).
[0257] [0257] In one embodiment of the invention, a HEPN domain comprises at least one RxxxxH motif comprising the sequence of R(N/H/K)X1X2X3H. In one embodiment of the invention, a HEPN domain comprises an RxxxxH motif comprising the sequence of R(N/H)X1X2X3H. In one embodiment of the invention, a HEPN domain comprises the sequence RIN/K)X1X2X3H. In certain embodiments, X1 is R, S, D, E, Q, N, G, Y, or H. In certain embodiments, X2 is IL, S, T, V, or L. In certain embodiments, X3 is L, F, N, Y, V, I, S, D, E or A.
[0258] [0258] CARF domains and consensus sequences for CARF domains have been described (see, for example, Makarova et al., Front Genet. 2014; 5:102). In certain embodiments, Csm6 comprises at least one CARF domain comprising a core domain comprising a six-strand Rossmann-like bend with the core of strands 5 and 6 forming a hook pin à. The main sequence conservation regions are associated with the 1-strand and the 4-strand of the main domain. In certain embodiments, the end of the l-strand is characterized by a polar residue, typically with an alcoholic side chain.
[0259] [0259] In certain embodiments, the Csm6 comprises at least one 6H domain (Niewoehner and Jinek, 2016). The 6H domain of the TtCsm6 polypeptide chain (residues 191 to 292) consists of five a-helices and forms a right-handed solenoid domain. Not being bound by theory, since some orthologs may not have a 6H domain, this domain is not required for the activity of the Csm6 protein of the present invention.
[0260] [0260] Csm6 has been shown to contribute to interference by functioning as an autonomous ribonuclease that degrades the invader's RNA transcripts. Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon binding of target RNA by the type III interference complex, the Casl10 subunit converts ATP to a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. Mutations in the CARF domain that abolish allosteric activation inhibit Csm6 activity in vivo and phenocopy-loss mutations of the Casl0 Palm domain of Csm6 (M. Kazlauskiene et al., 2017; eNiewoehner et al. 2017).
[0261] [0261] In certain example embodiments, the signal amplification CRISPR effector protein is activated when the activated CRISPR detection protein breaks an activation sequence. Activation sequences are described in more detail below. The activation sequence cleavage product activates a separate activity of the signal amplification CRISPR effector protein, such as an RNA nuclease activity. For example, Csm6, once activated, cleaves RNA indiscriminately similar to the side effect of Casl13 enzymes. Thus, in addition to effector modification detecting the reporter constructs, the activated signal amplification effector protein CRISPR also modifies the reporter constructs to further improve signal generation. In certain embodiments, Csm6e is activated when given in conjunction with another CRISPR enzyme (eg, Casl3). In certain embodiments, Csm6 can generate a synergistic effect when used in conjunction with Casl3, so that the collateral activity of Casl13 is greatly increased. Not being bound by theory, the Casl13 concentration can be greatly decreased in a test when Csm6 is also included in the test (eg point-of-care test). So, the addition of
[0262] [0262] In certain example embodiments, the one or more signal amplification effector proteins are selected in Table 6.
[0263] [0263] CRISPR effectors generally interact with additional components to modulate activity, and type VI-B CRISPR systems generally harbor the interference modulating proteins Csx27 and Csx28, and co-expression of Csx28 has been shown to increase the interference activity of the Csx27 and Csx28 interference modulators. Casl3b proteins in vivo. In certain exemplary embodiments, the one or more signal amplification CRISPR effector proteins comprise Csx28 or Csx27. TARGET AMPLIFICATION
[0264] [0264] In certain exemplary embodiments, target RNAs and/or DNAs may be amplified prior to activation of the CRISPR effector protein. Any suitable RNA or DNA amplification technique can be used. In certain exemplary embodiments, the RNA or DNA amplification is isothermal amplification. In certain exemplary embodiments, isothermal amplification can be nucleic acid sequence based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand shift amplification (SDA), amplification dependent helicase (HDA) or enzymatic amplification reaction (NEAR). In certain exemplary embodiments, non-isothermal amplification methods may be used, which include, but are not limited to, PCR, multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or branch amplification (RAM).
[0265] [0265] In certain example embodiments, RNA or DNA amplification is NASBA nucleic acid sequence-based amplification, which is initiated with reverse transcription of target RNA by a sequence-specific reverse primer to create an RNA/ DNA. RNase H is then used to degrade the RNA template, allowing a forward primer containing a promoter, such as the T7 promoter, to bind and initiate elongation of the complementary strand, generating a double-stranded DNA product. RNA polymerase promoter-mediated transcription of the DNA template creates copies of the target RNA sequence. Importantly, each of the new target RNAs can be detected by the chi RNAs, further improving the sensitivity of the assay. Binding of target RNAs by guide RNAs leads to activation of the CRISPR effector protein and the methods proceed as described above. The NASBA reaction has the added advantage that it can proceed under moderate isothermal conditions, for example approximately 41°C, making it suitable for systems and devices deployed for early and direct detection in the field and away from clinical laboratories.
[0266] [0266] In certain other exemplary embodiments, a recombinase polymerase (RPA) amplification reaction may be used to amplify target nucleic acids. RPA reactions employ recombinases that are capable of pairing sequence-specific primers with homologous sequence in duplex DNA.
[0267] [0267] Therefore, in certain exemplary embodiments, the systems disclosed herein may include amplification reagents. Different components or reagents useful for amplifying nucleic acids are described here. For example, an amplification reagent as described herein may include a buffer, such as a Tris buffer. A Tris buffer can be used at any concentration appropriate for the application or desired use, for example, including but not limited to a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 MM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 25 mM, 50 mM, 75 mM, 1 M or the like. One skilled in the art will be able to determine an appropriate concentration of a buffer, such as Tris, for use with the present invention.
[0268] [0268] A salt such as magnesium chloride (MgCl2), potassium chloride (KCl) or sodium chloride (NaCl) can be included in an amplification reaction such as PCR in order to improve amplification of acid fragments nucleic. Although the salt concentration depends on the particular reaction and application, in some embodiments, nucleic acid fragments of a specific size can produce optimal results at specific salt concentrations. Larger products may require altered salt concentrations, typically lower salt, to produce the desired results, while amplification of smaller products may produce better results at higher salt concentrations. One skilled in the art will understand that the presence and/or concentration of a salt, along with changing salt concentrations, can alter the stringency of a biological or chemical reaction, and therefore any salt can be used that provides the proper conditions for a reaction of the present invention and as described herein.
[0269] [0269] Other components of a biological or chemical reaction may include a cell lysis component to disrupt or lyse a cell for analysis of the materials contained therein. A cell lysis component may include, but is not limited to, a detergent, a salt as described above, such as NaCl, KCl, ammonium sulfate [(NHa4)2SOs], or others. Detergents that may be suitable for the invention may include Triton X*X-100, sodium dodecyl sulfate (SDS), CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate), ethyl trimethyl ammonium bromide, nonylphenoxypolyethoxyethanol (NP-40). Detergent concentrations may depend on the specific application and, in some cases, may be reaction specific. Amplification reactions may include dNTPs and nucleic acid primers used at any concentration appropriate for the invention, such as including but not limited to a concentration of 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM , 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM,
[0270] [0270] In some embodiments, amplification reagents as described herein may be suitable for use in warm start amplification. Hot start amplification can be beneficial in some embodiments to reduce or eliminate dimerization of adapter molecules or oligos, or to prevent unwanted amplification products or artifacts and achieve optimal amplification of the desired product. Many components described here for use in amplification can also be used in hot start amplification. In some embodiments, reagents or components suitable for use with hot-start amplification may be used in place of one or more of the components of the composition, as appropriate. For example, a polymerase or other reagent can be used that exhibits a desired activity at a specific temperature or other reaction condition. In some embodiments, reagents that are designed or optimized for use in hot-start amplification can be used, for example, a polymerase can be activated after transposition or after reaching a specific temperature. Such polymerases may be antibody-based or apatamer-based. Polymerases as described herein are known in the art. Examples of such reagents may include, but are not limited to, hot-start polymerases, hot-start dNTPs, and photoaging dNTPs. Such reagents are known and available in the art. One skilled in the art will be able to determine optimal temperatures as appropriate for individual reagents.
[0271] [0271] Nucleic acid amplification can be performed using specific thermal cycling machines or equipment and can be performed in single reactions or in bulk, so that any desired number of reactions can be carried out simultaneously. In some embodiments, amplification can be performed using microfluidic or robotic devices, or it can be performed using manual change in temperatures to achieve the desired amplification. In some embodiments, optimization can be performed to obtain optimal reaction conditions for the specific application or materials. One skilled in the art will understand and be able to optimize the reaction conditions to obtain sufficient amplification.
[0272] [0272] In certain embodiments, detection of DNA with the methods or systems of the invention requires transcription of the (amplified) DNA into RNA prior to detection. DETECTABLE POSITIVE SIGNAL INTENSIFICATION
[0273] [0273] In certain exemplary embodiments, other modifications may be introduced that further amplify the detectable positive signal. For example, collateral activation of activated CRISPR effector protein can be used to generate a secondary target or an additional guide sequence, or both. In an example embodiment, the reaction solution would contain a secondary target that is spiked in high concentration. The secondary target may be distinct from the primary target (ie, the target the assay is designed to detect) and, in certain cases, may be common across all reaction volumes. A secondary guide sequence for the secondary target may be protected, for example, by a secondary structural feature, such as a hairpin with an RNA loop, and unable to bind the second target or the CRISPR effector protein. Cleavage of the protecting group by an activated CRISPR effector protein (i.e., after activation by complex formation with the primary target(s) in solution) and formation of a complex with free CRISPR effector protein in solution and activation of the spike on the secondary target. In certain other example embodiments, a similar concept is used with free guide sequence for a secondary target and a protected secondary target. Cleavage of a protective group from the secondary target would allow formation of additional CRISPR effector protein, guide sequence, secondary target sequence. In yet another example embodiment, activation of the CRISPR effector protein by the primary target(s) can be used to cleave a shielded or circularized primer, which would then be released to perform an isothermal amplification reaction, such as those disclosed herein, in a model for the secondary target sequence, secondary target, or both. Subsequent transcription “from this amplified template would produce more secondary guide sequence and/or secondary target sequence, followed by further activation of the CRISPR effector protein. EXAMPLE METHODS AND TESTS
[0274] [0274] The low cost and adaptability of the assay platform lends itself to a number of applications, including (i) general RNA/DNA quantification, (ii) rapid and multiplexed detection of RNA/DNA expression, and (iii) sensitive detection of target nucleic acids, peptides in clinical and environmental samples. Furthermore, the systems disclosed herein may be adapted for detecting transcripts within biological settings, such as cells. Given the highly specific nature of the CRISPR effectors described herein, it may be possible to screen for allelic-specific expression of disease-associated transcripts or mutations in living cells.
[0275] [0275] In certain example embodiments, a single guide sequence specific to a single target is placed in separate volumes. Each volume can then receive a different sample or aliquot of the same sample. In certain example embodiments, multiple guide sequences each for a separate target can be placed in a single well, so that multiple targets can be tracked in a different well. To detect multiple guide RNAs in a single volume, in certain example modalities, multiple effector proteins with different specificities can be used. For example, different orthologs with different sequence specificities can be used. For example, one ortholog may preferentially cut A, while others preferentially cut C, G, U/T. Therefore, masking constructs can be generated that are all or comprise a substantial portion of a single nucleotide, each with a different fluorophore that can be detected at different wavelengths. In this way, up to four different targets can be tracked in a single individual discrete volume. In certain exemplary embodiments, different orthologs of the same CRISPR effector protein class may be used,
[0276] [0276] As demonstrated here, CRISPR effector systems are capable of detecting atmolar concentrations of target molecules. See, for example, FIGs. 13, 14, 19, 22 and Examples described below. Due to the sensitivity of said systems, various applications that require rapid and sensitive detection can benefit from the modalities disclosed in this document and are contemplated as being within the scope of the invention. Sample assays and applications are described in more detail below. MICROBIAL APPLICATIONS
[0277] [0277] In certain exemplary embodiments, the systems, devices, and methods disclosed herein are directed toward detecting the presence of one or more microbial agents in a sample, such as a biological sample obtained from a subject. In certain exemplary embodiments, the microbe can be a bacterium, a fungus, a yeast, a protozoan, a parasite, or a virus. Accordingly, the methods disclosed herein can be adapted for use in other methods (or in combination) with other methods that require rapid identification of microbial species, monitoring the presence of microbial proteins (antigens), antibodies, antibody genes, detection of certain phenotypes (eg, bacterial resistance), monitoring of disease progression and/or outbreak, and antibiotic tracking. Due to the rapid and sensitive diagnostic capabilities of the modalities disclosed herein, detection of microbe species type, down to a single nucleotide difference, and the ability to be implanted as a POC device, the modalities disclosed herein can be used as guide therapeutic regimens. , such as a selection of the appropriate antibiotic or antiviral. The modalities disclosed in this document can also be used to screen environmental samples (air, water, surfaces, food, etc.) for the presence of microbial contamination.
[0278] [0278] A method for identifying microbial species, such as bacterial, viral, fungal, yeast, or parasite species, or the like is disclosed. Particular embodiments disclosed in this document describe methods and systems that will identify and distinguish microbial species in a single sample or in multiple samples, allowing the recognition of many different microbes. The present methods allow the detection of pathogens and the distinction between two or more species of one or more organisms, for example, bacteria, viruses, yeasts, protozoa and fungi or a combination thereof, in a biological or environmental sample, detecting the presence of a target nucleic acid sequence in the sample. A positive signal obtained from the sample indicates the presence of the microbe. Multiple microbes can be identified simultaneously using the methods and systems of the invention, employing the use of more than one effective protein, where each effector protein targets a specific microbial target sequence. In this way, a multilevel analysis can be performed for a particular subject in which any number of microbes can be detected at the same time. In some embodiments, simultaneous detection of multiple microbes can be accomplished using a set of probes that can identify one or more microbial species.
[0279] [0279] Multiplexed sample analysis allows large-scale sample detection, reducing analysis time and cost. However, multiplex analyzes are often limited by the availability of a biological sample. In accordance with the invention, however, alternatives to multiplex analysis can be performed so that multiple effector proteins can be added to a single sample and each mask construct can be combined with a separate quencher dye. In this case, positive signals from each extinguishing dye can be obtained separately for multiple detection in a single sample.
[0280] [0280] Disclosed herein are methods for distinguishing between two or more species of one or more organisms in a sample. The methods are also capable of detecting one or more species of one or more organisms in a sample.
[0281] [0281] In some embodiments, a method is provided for detecting microbes in samples comprising dispensing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system as described herein; incubating the sample or set of samples under conditions sufficient to allow binding of one or more guide RNAs to one or more specific microbial targets; activating the CRISPR effector protein by binding one or more guide RNAs to one or more target molecules, wherein activation of the CRISPR effector protein results in the modification of the RNA-based mask construct such that a detectable positive signal is generated; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates the presence of one or more target molecules in the sample.
[0282] [0282] In some embodiments, one or more identified target sequences can be detected using guide RNAs that are specific and bind to the target sequence, as described here. The systems and methods of the present invention can distinguish even single nucleotide polymorphisms present between different microbial species and therefore the use of various guide RNAs in accordance with the invention can further expand or improve the number of target sequences that can be used. to distinguish between species. For example, in some embodiments, the one or more guide RNAs can distinguish between microbes in the species, genus, family, order, class, phylum, kingdom, or phenotype, or a combination thereof.
[0283] [0283] In certain exemplary embodiments, the devices, systems, and methods disclosed herein may be used to distinguish various microbial species in a sample. In certain exemplary embodiments, identification may be based on ribosomal RNA sequences, including the 16S, 23S, and 58 subunits. Methods for identifying relevant rRNA sequences are disclosed in the U.S. Patent Application Publication.
[0284] [0284] In certain example modalities, a method or diagnosis is designed to screen microbes through multiple phylogenetic and/or phenotypic levels at the same time. For example, the method or diagnosis may comprise the use of multiple CRISPR systems with different guide RNAs. A first set of guide RNAs can distinguish, for example, between mycobacteria, gram-positive and gram-negative bacteria. These general classes can be further subdivided. For example, guide RNAs can be designed and used in the method or diagnosis that distinguishes enteric and non-enteric in gram-negative bacteria. A second set of guide RNAs can be designed to distinguish microbes at the genus or species level. Thus, a matrix can be produced identifying all mycobacteria, gram-positive and gram-negative (divided into enteric and non-enteric) with each genus of bacterial species identified in a given sample that fall into one of these classes. The foregoing is for example purposes only. Other means of classifying other types of microbes are also contemplated and would follow the general structure described above.
[0285] [0285] In certain exemplary embodiments, the devices, systems, and methods disclosed herein may be used to screen for microbial genes of interest, for example, antibiotic and/or antiviral resistance genes. Chi RNAs can be designed to distinguish between known genes of interest. Samples, including clinical samples, can then be screened using the modalities disclosed herein for detecting such genes. The ability to screen for drug resistance in POC would be of tremendous benefit in selecting an appropriate treatment regimen. In certain exemplary embodiments, the antibiotic resistance genes are carbapenemases including KPC, NDM1, CTX-M15, OXA-48. Other antibiotic resistance genes are known and can be found, for example, in the comprehensive antibiotic resistance database (Jia et al. “CARD 2017: expansion and model-centric curation of the Comprehensive Antibiotic Resistance Database.” Nucleic Acids Research, 45, D566-573).
[0286] [0286] Ribavirin is an effective antiviral that targets several RNA viruses. Several clinically important viruses have evolved resistance to ribavirin, including foot-and-mouth disease virus doi: 10.1128/JVI.03594-13; polio virus ( Pfeifer and Kirkegaard. PNAS, 100 (12): 7289-7294, 2003 ); and hepatitis C virus ( Pfeiffer and Kirkegaard, J. Virol. 79(4):2346-2355, 2005 ). Several other persistent RNA viruses, such as hepatitis and HIV, have developed resistance to existing antiviral drugs: hepatitis B virus (lamivudine, tenofovir, entecavir) doi: 10/1002/hep22900; hepatitis C virus (telaprevir, BILN2061, ITMN-191, SCh6, boceprevir, AG-021541, ACH-806) doi: 10.1002/hep.22549; and HIV (many drug resistance mutations) hivb.standford.edu. The modalities disclosed in this document can be used to detect these variants among others.
[0287] [0287] In addition to drug resistance, there are several clinically relevant mutations that can be detected with the modalities disclosed herein, such as persistent versus acute infection in LCMV (doi:
[0288] [0288] As described elsewhere herein, closely related microbial species (eg, having only a single nucleotide difference in a given target sequence) can be distinguished by introducing a synthetic mismatch into the gRNA.
[0289] [0289] In particular embodiments, a set of guide RNAs is designed that can identify, for example, all microbial species within a defined set of microbes. Such methods are described in certain exemplary embodiments; methods for generating guide RNAs as described herein can be compared to the methods disclosed in WO 2017/040316, incorporated herein by reference. As described in WO 2017040316, a set coverage solution can identify the minimum number of target sequence probes or chi RNAs needed to cover an entire target sequence or a set of target sequences, e.g. a set of —“genomic” sequences .
[0290] [0290] In contrast, the modalities disclosed in this document are aimed at detecting longer probe or guide RNA lengths, for example in the range of 70 bp to 200 bp that are suitable for hybrid selection sequencing. Furthermore, the methods disclosed herein can be applied to adopt a pan-target sequence approach capable of defining a probe or defining RNA sets that can identify and facilitate sequencing and detection of all species and/or strain sequences in a large and/or variable target sequence set. For example, the methods disclosed herein can be used to identify all variants of a given virus or several different viruses in a single assay. Furthermore, the method disclosed in this document treats each element of the "universe" in the ensemble coverage problem as being one nucleotide of a target sequence, and each element is considered "covered" as long as a probe or guide RNA binds to some segment. of a target genome that includes the element.
[0291] [0291] The ability to detect multiple transcriptional abundances may allow the generation of unique microbial signatures indicative of a specific phenotype. Various machine learning techniques can be used to derive gene signatures. Therefore, guide RNAs from CRISPR systems can be used to identify and/or quantify relative levels of biomarkers defined by the gene signature in order to detect certain phenotypes. In certain exemplary embodiments, the gene signature indicates susceptibility to an antibiotic, resistance to an antibiotic, or a combination thereof.
[0292] [0292] In one aspect of the invention, a method comprises detecting one or more pathogens. In this way, differentiation between infection of a subject by individual microbes can be obtained. In some modalities, this differentiation may allow detection or diagnosis by a clinician of specific diseases, for example, different variants of a disease. Preferably, the pathogen sequence is a pathogen genome or a fragment thereof. The method may further comprise determining the evolution of the pathogen. Determining pathogen evolution may comprise identifying pathogen mutations, for example,
[0293] [0293] In some embodiments, a CRISPR system or methods of using it, as described here, may be used to determine the course of a pathogen outbreak. The method may comprise detecting one or more target sequences from a plurality of samples from one or more subjects, wherein the target sequence is a sequence from a microbe causing the outbreaks. This method may further comprise determining a pathogen transmission pattern or a mechanism involved in a disease outbreak caused by a pathogen.
[0294] [0294] The pattern of pathogen transmission may comprise continued new transmissions from the natural reservoir of the pathogen or subject-to-subject transmissions (eg, human-to-human transmission) after a single transmission from the natural reservoir or a mixture of both. In one embodiment, transmission of the pathogen may be bacterial or viral transmission; in that case, the target sequence is preferably a microbial genome or fragments thereof. In one embodiment, the pattern of pathogen transmission is the initial pattern of pathogen transmission, that is, at the beginning of the pathogen outbreak. Determining the pattern of pathogen transmission early in the outbreak increases the likelihood of stopping the outbreak as early as possible, thus reducing the possibility of local and international spread.
[0295] [0295] Determining the pattern of pathogen transmission may comprise detecting a sequence of pathogens in accordance with the methods described herein. Determining the pattern of pathogen transmission may further comprise detecting shared intra-host variations of the pathogen sequence between subjects and determining whether the shared intra-host variations show temporal patterns. Patterns in observed intra-host and inter-host variation provide important information about transmission and epidemiology (Gire, et al., 2014).
[0296] [0296] The detection of shared intra-host variations among subjects that show temporal patterns is an indication of transmission links between subjects (in particular between humans) because it can be explained by infection from multiple sources (superinfection), recurrent mutations contamination of the sample (with or without balancing selection to reinforce mutations) or the co-transmission of slightly divergent viruses that arose by mutation earlier in the chain of transmission (Park, et al., Cell 161(7):1516-1526, 2015). Detection of intra-host variations shared between subjects may comprise detecting intra-host variants located at common single nucleotide polymorphism (SNP) positions. The positive detection of intra-host variants located in common positions (SNP) is indicative of superinfection and contamination as primary explanations for the intra-host variants. Superinfection and contamination can be divided based on the frequency of the SNP that appears as inter-host variants (Park, et al., 2015). Otherwise, superinfection and contamination can be ruled out. In the latter case, the detection of intra-host shared variations between subjects may also include evaluating the frequencies of synonymous and non-synonymous variants and comparing the frequency of synonymous and non-synonymous variants with each other. A non-synonymous mutation is a mutation that alters the amino acid of the protein, likely resulting in a biological change in the microbe subject to natural selection. Synonymous substitution does not change an amino acid sequence. Equal frequency of synonymous and non-synonymous variants is indicative that intra-host variants evolved neutrally. If the frequencies of synonymous and non-synonymous variants are divergent, it is likely that intra-host variants are maintained by balanced selection. If the frequencies of synonymous and non-synonymous variants are low, this is indicative of a recurrent mutation. If the frequencies of synonymous and non-synonymous variants are high, this is indicative of co-transmission (Park, et al., 2015).
[0297] [0297] Like the Ebola virus, the Lassa virus (LASV) can cause hemorrhagic fever with high mortality rates. Andersen et al. generated a genomic catalog of nearly 200 LASV sequences from clinical samples and rodent reservoirs (Andersen, et al., Cell Volume 162, Issue 4, p 738-750, 13 August 2015). Andersen et al. show that while the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections primarily result from reservoir-to-human infections. Andersen et al. elucidated the spread of LASV in West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translation efficiency. The method may further comprise phylogenetically comparing a first pathogen sequence with a second pathogen sequence and determining whether a phylogenetic link exists between the first and second pathogen sequences. The second pathogen sequence may be a previous reference sequence. If there is a phylogenetic link, the method may further comprise rooting the phylogeny from the first pathogen sequence to the second pathogen sequence. Thus, it is possible to construct the lineage of the first pathogen sequence (Park, et al., 2015).
[0298] [0298] The method may further comprise determining whether the mutations are deleterious or adaptive. Deleterious mutations are indicative of viruses impaired in transmission and dead-end infections, therefore normally present only in an individual. Mutations unique to a subject are those that occur in the outer branches of the phylogenetic tree, while mutations in the inner branches are those present in multiple samples (ie, in multiple subjects). Higher non-synonymous replacement rate is a feature of the outer branches of the phylogenetic tree (Park, et al., 2015).
[0299] [0299] In the inner branches of the phylogenetic tree, selection had more opportunity to filter out deleterious mutants. Internal branches, by definition, have produced multiple descendant lines and are therefore less likely to include mutations with fitness costs. Thus, lower non-synonymous replacement rate is indicative of internal ramifications (Park, et al., 2015).
[0300] [0300] Synonymous mutations, which are likely to have less impact on fitness, occurred at more comparable frequencies in the inner and outer branches (Park, et al., 2015).
[0301] [0301] By analyzing the sequenced target sequence, such as viral genomes, it is possible to discover the mechanisms responsible for the severity of the epidemic episode, such as during the Ebola outbreak in 2014. For example, Gire et al. made a phylogenetic comparison of the genomes from the 2014 outbreak with all 20 genomes from previous outbreaks, which suggests that the 2014 West African virus likely spread from central Africa in the last decade. Phylogeny rooting using divergence with other ebolavirus genomes was problematic (6, 13). However, rooting the tree in the oldest outbreak revealed a strong correlation between sample date and root-to-tip distance, with a replacement rate of 8 x 10-4 per site per year (13). This suggests that the lineages of the three most recent outbreaks diverged from a common ancestor at approximately the same time, around 2004, which supports the hypothesis that each outbreak represents an independent zoonotic event from the same genetically diverse viral population in its natural reservoir. They also found that the 2014 EBOV outbreak could be caused by a single transmission from the natural reservoir, followed by human-to-human transmission during the outbreak. Their results also suggested that the epidemic episode in Sierra Leone may result from the introduction of two genetically distinct viruses from Guinea at the same time (Gire, et al., 2014).
[0302] [0302] It was also possible to determine how the Lassa virus spread from its point of origin, in particular thanks to human-to-human transmission; and it was even possible to retrace the history of this propagation 400 years ago (Andersen, et al., Cell 162(4):738-50, 2015).
[0303] [0303] Regarding the work required during the 2013-2015 EBOV outbreak and the difficulties encountered by medical staff at the outbreak site, and more generally, the method of the invention makes it possible to perform sequencing using fewer selected probes, so that sequencing can be accelerated, thus reducing the time required from taking samples to obtaining results. In addition, kits and systems can be designed for use in the field so that a patient's diagnosis can be made quickly without the need to ship or ship samples to another part of the country or the world.
[0304] [0304] In any method described above, sequencing the target sequence or fragment thereof may use any of the sequencing procedures described above. Furthermore, sequencing the target sequence or fragment thereof can be near real-time sequencing. The sequencing of the target sequence or fragment thereof can be performed according to previously described methods (Experimental Procedures: Matranga et al., 2014; and Gire, et al., 2014). Sequencing the target sequence or fragment thereof may comprise parallel sequencing of a plurality of target sequences. Sequencing the target sequence or fragment thereof may comprise Illumina sequencing.
[0305] [0305] Analysis of the target sequence or fragment thereof that hybridizes to one or more of the selected probes may be an identification analysis, in which the hybridization of a selected probe to the target sequence or a fragment thereof indicates the presence of the sequence target in the sample.
[0306] [0306] Currently, the primary diagnosis is based on the symptoms a patient presents. However, several diseases can share identical symptoms, so the diagnosis relies heavily on statistics. For example, malaria triggers flu-like symptoms: headache, fever, tremors, joint pain, vomiting, hemolytic anemia, jaundice, hemoglobin in the urine, retinal damage, and seizures. These symptoms are also common for septicemia, gastroenteritis, and viral illnesses. Among the latter, Ebola hemorrhagic fever has the following symptoms: fever, sore throat, muscle pain, headache, vomiting, diarrhea, rash, decreased liver and kidney function, internal and external bleeding.
[0307] [0307] When a patient is presented to a medical facility, for example in tropical Africa, the basic diagnosis ends up with malaria, because statistically, malaria is the most likely disease in that region of Africa. Consequently, the patient is treated for malaria, although they may not actually have contracted the disease and end up not being treated properly. This lack of correct treatment can be fatal, especially when the disease contracted by the patient has a rapid evolution. It may be too late for the medical team to realize that the treatment given to the patient is ineffective and arrive at the correct diagnosis and administer the appropriate treatment to the patient.
[0308] [0308] The method of the invention provides a solution to this situation. In fact, as the number of guide RNAs can be drastically reduced, this makes it possible to provide, on a single chip, selected probes divided into groups, each group being specific for a disease, so that a plurality of diseases, such as viral infection, can be be diagnosed at the same time. Thanks to the invention, more than 3 diseases can be diagnosed on a single chip, preferably more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 diseases at the same time, preferably diseases that occur most commonly in the population of a given geographic area. As each group of probes selected is specific to one of the diagnosed diseases, a more accurate diagnosis can be made, decreasing the risk of administering the wrong treatment to the patient.
[0309] [0309] In other cases, an illness such as a viral infection may occur without any symptoms or cause symptoms, but they disappeared before the patient was presented to the medical team. In these cases, the patient does not seek medical attention or the diagnosis is complicated due to the absence of symptoms on the day of presentation.
[0310] [0310] The present invention can also be used in conjunction with other methods of diagnosing diseases, identifying pathogens and optimizing treatment based on the detection of nucleic acids such as mRNA in raw and unpurified samples.
[0311] [0311] The method of the invention also provides a powerful tool to resolve this situation. In fact, since a plurality of selected groups of guide RNAs, each group being specific for one of the most common diseases that occur in the population of a certain area, is composed of a single diagnosis, the medical team only needs to contact a biological sample taken from the patient with the chip. The chip reading reveals the diseases that the patient has contracted.
[0312] [0312] In some cases, the patient is presented to the medical team for diagnosis of specific symptoms. The method of the invention makes it possible not only to identify which disease causes these symptoms, but at the same time to determine whether the patient suffers from another disease of which he was not aware.
[0313] [0313] This information can be of utmost importance when looking for the mechanisms of an outbreak. Indeed, groups of patients with identical viruses also show temporal patterns, suggesting a subject-to-subject transmission link.
[0314] [0314] In certain exemplary embodiments, the CRISPR systems disclosed herein may be used to screen for microbial genetic disorders. Such methods can be useful, for example, for mapping microbial pathways and functional networks. Microbial cells can be genetically modified and then screened under different experimental conditions. As described above, the modalities disclosed herein can screen multiple target molecules in a single sample or a single target in a single discrete volume in a multiplex manner. Genetically modified microbes can be modified to include a nucleic acid barcode sequence that identifies the specific genetic modification performed by a specific microbial cell or population of microbial cells. A barcode is a short sequence of nucleotides (eg DNA, RNA or combinations thereof) that is used as an identifier. A nucleic acid barcode can be 4-100 nucleotides in length and be single-stranded or double-stranded. Methods for identifying cells with barcodes are known in the art. Therefore, the guide RNAs from the CRISPR effector systems described herein can be used to detect the barcode. The detection of the positive detectable signal indicates the presence of a specific genetic modification in the sample. The methods disclosed herein can be combined with other methods to detect complementary genotypes or phenotypic readouts indicating the effect of the genetic modification under the "experimental conditions tested." Genetic modifications to be screened may include, but are not limited to, a gene knock-in, a gene knock-out, inversions, translocations, transpositions, or one or more insertions, deletions, substitutions, mutations, or nucleic acid additions that encode an epitope with a functional consequence, such as alteration of stability or detection of proteins. Similarly, the methods described herein can be used in synthetic biology applications to screen the functionality of specific arrays of gene regulatory elements and gene expression modules.
[0315] [0315] In certain exemplary embodiments, the methods may be used to track hypomorphs. The generation of hypomorphs and their use in the identification of the main bacterial functional genes and in the identification of new antibiotic therapies, as disclosed in PCT/US2016/060730, entitled “Multiplex High-Resolution Detection of Micro-organism Strains, Related Kits, Diagnostic Methods and Screening Assays”, filed November 4, 2016, which is incorporated herein by reference.
[0316] [0316] Different experimental conditions may include exposure of microbial cells to different chemical agents, combinations of chemical agents, different concentrations of chemical agents or combinations of chemical agents, different durations of exposure to chemical agents or combinations of chemical agents, different parameters physical or both. In certain exemplary embodiments, the chemical agent is an antibiotic or antiviral. Different physical parameters to be tracked may include different temperatures, atmospheric pressures, different atmospheric and non-atmospheric concentrations of gases, different pH levels, different compositions of culture media or a combination thereof.
[0317] [0317] The methods disclosed herein may also be used to screen environmental samples for contaminants by detecting the presence of target nucleic acid or polypeptides. For example, in some embodiments, the invention provides a method for detecting microbes, comprising: exposing a CRISPR system as described herein to a sample; activating an RNA effector protein via binding of one or more guide RNAs to one or more microbial-specific target RNAs or to one or more trigger RNAs so that a detectable positive signal is produced. The positive signal can be detected and is indicative of the presence of one or more microbes in the sample. In some embodiments, the CRISPR system may be on a substrate as described herein, and the substrate may be exposed to the sample. In other embodiments, the same CRISPR system and/or a different CRISPR system can be applied to several different locations on the substrate. In other embodiments, the different CRISPR system may detect a different microbe at each location. As described in more detail above, a substrate may be a substrate of flexible materials, for example, including but not limited to a paper substrate, a fabric substrate or a polymer-based flexible substrate.
[0318] [0318] In accordance with the invention, the substrate can be passively exposed to the sample by temporarily immersing the substrate in a fluid to be sampled, applying a fluid to be tested to the substrate, or by contacting a surface to be tested with the substrate. . Any means of introducing the sample to the substrate may be used as appropriate.
[0319] [0319] As described herein, a sample for use with the invention can be a biological or environmental sample, such as a food sample (fresh fruits or vegetables, meats), a beverage sample, a paper surface, a fabric surface , a metal surface, a wooden surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saline water sample, exposure to atmospheric air or to another gas sample or a combination of them.
[0320] [0320] In some modalities, check the contamination of food by bacteria, such as E. coli, in restaurants or other food suppliers; food surfaces; testing the water for pathogens such as Salmonella, Campylobacter, or E. coli; and also check food quality by manufacturers and regulators to determine the purity of meat sources; identify air contamination with pathogens such as legionella; check whether the beer is contaminated or spoiled by pathogens such as Pediococcus and Lactobacillus; contaminate pasteurized or unpasteurized cheese with bacteria or fungi during manufacture.
[0321] [0321] A microbe according to the invention may be a pathogenic microbe or a microbe that results in spoilage of food or consumables. A pathogenic microbe can be pathogenic or undesirable to humans, animals or plants. For human or animal purposes, a microbe can cause disease or result in disease. Animal or veterinary applications of the present invention can identify animals infected with a microbe. For example, the methods and systems of the invention can identify companion animals with pathogens, including but not limited to kennel cough, rabies virus and heartworms. In other embodiments, the methods and systems of the invention can be used for parentage testing for breeding purposes. A plant microbe can result in damage or disease to a plant, reduction in yield, or change in traits such as color, taste, consistency, and odor. For purposes of contamination of food or consumables, a microbe can adversely affect the taste, odor, color, consistency or other commercial properties of the food or consumable product. In certain exemplary embodiments, the microbe is a bacterial species. The bacteria can be a psychrotroph, a coliform, a lactic acid bacteria or a spore-forming bacteria. In certain exemplary embodiments, the bacterium can be any bacterial species that causes disease or illness Or results in an undesirable product or trait. Bacteria according to the invention can be pathogenic for humans, animals or plants.
[0322] [0322] Samples suitable for use in the methods disclosed herein include any conventional biological sample obtained from an organism or part thereof, such as a plant, animal, bacterium and the like.
[0323] [0323] A sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy sample, such as a tumor biopsy) or can include a cell (either a primary cell or a cultured cell) or a conditioned medium by any cell, tissue or organ. Examples of samples include, without limitation, cells, cell lysates, blood smears, cytocentrifuge preparations, cytology smears, body fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.) , tissue biopsies (eg, tumor biopsies), fine needle aspirates, and/or tissue sections (eg, cryostat tissue sections and/or paraffin-embedded tissue sections). In other examples, the sample includes circulating tumor cells (which can be identified by cell surface markers). In particular examples, samples are used directly (e.g. fresh or frozen) or may be manipulated prior to use, e.g. by fixation (e.g. using formalin) and/or wax embedding (such as tissue samples soaked in paraffin and formalin fixed (FFPE)). It will be appreciated that any method for obtaining tissue from a subject can be used and that the selection of the method used will depend on a number of factors, such as tissue type, subject age, or procedures available to the practitioner. Standard techniques for acquiring such samples are available in the art. See, for example, Schluger et al., J. Exp. Med. 176:1327-33 (1992); Bigby et al., Am. Rev. Respir. Dis. 133:515-18 (1986); Kovacs et al., NEJM 318:589-93 (1988); and Ognibene et al., Am. Rev. Respir. Dis. 129:929-32 (1984).
[0324] [0324] In other embodiments, a sample can be an environmental sample such as water, soil, or a surface such as an industrial or medical surface. In some embodiments, methods such as those disclosed in U.S. patent publication 2013/0190196 can be applied to the detection of nucleic acid signatures, specifically RNA levels, directly from crude cell samples with a high degree of sensitivity and specificity. The specific sequences for each pathogen of interest can be identified or selected by comparing the coding sequences of the pathogen of interest with all the coding sequences in other organisms by the BLAST software.
[0325] [0325] Various embodiments of the present disclosure involve the use of procedures and approaches known in the art to successfully fractionate clinical blood samples. See, for example, the procedure described in Han Wei Hou et al., Microfluidic Devices for Blood
[0326] [0326] In addition, various embodiments of the present disclosure involve using procedures and approaches known in the art to successfully isolate pathogens from whole blood using spiral microchannel, as described in HHan Wei Hou et al., Pathogen Isolation from Whole Blood Using Spiral Microchannel, Case No. 15995JIR, Massachusetts Institute of Technology, manuscript in preparation, the disclosure of which is incorporated herein by reference in its entirety.
[0327] [0327] Due to the increased sensitivity of the modalities disclosed herein, in certain example modalities, the assays and methods may be performed on raw samples or samples in which the target molecules to be detected are no longer fractionated or purified from the sample.
[0328] [0328] The modality disclosed herein can be used to detect a number of different microbes. The term microbe as used herein includes bacteria, fungi, protozoa, parasites and viruses.
[0329] [0329] The following is a list of examples of the types of microbes that can be detected using the modalities disclosed in this document. In certain exemplary embodiments, the microbe is a bacterium. Examples of bacteria that can be detected according to the disclosed methods include, without limitation, any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma bPhagocytophilum, Anaplasma marginale Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sP., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila bpneumoniae, Chlamydophila psittaci, Citrobacter sp.
[0330] [0330] In certain exemplary embodiments, the microbe is a fungus or a species of fungus. Examples of fungi that can be detected according to the disclosed methods include, without limitation, any one or more of (or any combination of), Aspergillus, Blastomyces, Candidiasis, Coceidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp. Histoplasma sp. (as Histoplasma capsulatum) Pneumocystis sp. (such as Pneumocystis jirovecii) Stachybotrys (such as Stachybotrys chartarum), Mucroymcosis, Sporothrix, fungal eye infections, ringworm, Exserohilum, Cladosporium.
[0331] [0331] In certain example embodiments, the fungus is a yeast. Examples of yeasts that can be detected according to the disclosed methods include, without limitation, one or more of (or any combination of) Aspergillus species (such as "Aspergillus fumigatus, Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp. (such as Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii and Cryptococcus albidus), a Geotrichum species, Saccharomyces, a Hansenula species, a Candida species (such as Candida albicans), a Kluyveromyces species, a Debaryomyces species, a Pichia species, or combinations thereof. In certain exemplary embodiments, the fungus is a mold. Examples of molds include, but are not limited to, a Penicillium species, a Cladosporium species, a Byssochlamys species, or a combination thereof.
[0332] [0332] In certain exemplary embodiments, the microbe is a protozoan. Examples of protozoa that can be detected according to the disclosed methods and devices include, without limitation, any one or more of (or any combination of), Euglenozoa, Heterolobosea, Diplomonadida, Amoebozoa, Blastocystic and Apicomplexa. Examples of euglenoza include, but are not limited to, Trypanosoma cruzi (Chagas disease), T. brucei gambiense, T. brucei rhodesiense, Leishmania braziliensis, L. infantum, L. mexicana, L. major, L. tropica, and L. donovani. Examples of euglenoza include, but are not limited to, Naegleria fowleri. Examples of diplomonadid include, but are not limited to, Giardia intestinalis (G. lamblia, G. duodenalis). Examples of Amoebozoa include, but are not limited to, Acanthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica. Exemplary Blastocystis include, but are not limited to, Blastocystic hominis. Examples of apicomplexa include, but are not limited to, Babesia microti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and Toxoplasma gondii, Babesia microti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodium falciparum , P. vivax, P. ovale, P. malariae, and Toxoplasma gondii.
[0333] [0333] In certain exemplary embodiments, the microbe is a parasite. Examples of parasites that can be detected according to the disclosed methods include, without limitation, one or more of (or any combination of), an Onchocerca species and a Plasmodium species.
[0334] [0334] In certain example embodiments, the systems, devices and methods disclosed herein are directed at detecting viruses in a sample. The modalities disclosed herein can be used to detect viral infection (e.g., of a subject or plant) or determination of a viral strain, including viral strains that differ by a single nucleotide polymorphism. The virus can be a DNA virus, a virus of
[0335] [0335] In certain exemplary embodiments, the virus may be a plant virus selected from the group comprising tobacco mosaic virus (TMV), tomato wilt virus (TSWV), or cucumber mosaic virus ( CMV), potato virus Y (PVY), RT virus, cauliflower mosaic virus (CaMV), plum virus (PPV), Brome mosaic virus (BMV), potato virus X (PVX), Citrus tristeza virus (CTV), barley yellow dwarf virus (BYDV), potato leaf virus (PLRV), tomato jam virus (TBSV), tungro rice spherical virus (RTSV), yellow rice mosquito virus (RYMV), hoja blanca rice virus (RHBV), rayado fino maize virus (MRFV), maize dwarf mosaic virus (MDMV), sugar cane mosaic virus (SCMV), sugar cane mosaic virus (SCMV), Sweet Potato Mosquito Virus (SPFMV), Sweet Potato Sunken Vein Closterovirus (SPSVV), Vine Fan Leaf Virus (GFLV), Vine Virus A (GVA), Vine Virus B (GVB) ), come over vine leafroll-associated virus (GFkV), vine leafroll-associated virus-1, -2 and -3 (GLRaV-1, -2 and -3), Arabis mosaic virus (ArMV) or stem-bite-associated virus Rupestris (RSPaV). In a preferred embodiment, the target RNA molecule is part of said pathogen or transcribed from a DNA molecule of said pathogen. For example, the target sequence may be comprised in the genome of an RNA virus. It is further preferred that the CRISPR effector protein hydrolyses said target RNA molecule of said pathogen in said plant if said pathogen infects or has infected said plant. Therefore, it is preferable that the CRISPR system is able to cleave the target RNA molecule of the plant pathogen when the CRISPR system (or parts necessary for its completion) is applied therapeutically, i.e. after infection, or prophylactically i.e. before infection.
[0336] [0336] In certain example embodiments, the virus may be a retrovirus. Examples of retroviruses that can be detected using the modalities disclosed herein include one or more of or any combination of viruses of the genus Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus, Spumavirus or the Family Metaviridae, Pseudoviridae and HIV Retroviridae), Hepadnaviridae ( including hepatitis B virus) and Caulimoviridae (including cauliflower mosaic virus).
[0337] [0337] In certain embodiments of the example, the virus is a DNA virus. Examples of DNA viruses that can be detected using the modalities disclosed herein include one or more of (or any combination of) viruses from the Family Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus and Varicella Zoster virus), Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fever virus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae , Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, BK virus), Poxviridae (including cowpox and smallpox, Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus, among others. In some embodiments, a method of diagnosing a species-specific bacterial infection in a subject suspected of having a bacterial infection is described as obtaining a sample comprising bacterial ribosomal ribonucleic acid from the subject; contact the sample with one or more of the probes described and detect hybridization between the bacterial ribosomal ribonucleic acid sequence present in the sample and the probe, where detection of hybridization indicates that the subject is infected with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa , Staphylococcus aureus, Acinetobacter baumannii, Candida albicans, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Staphylococcus agalactiae or Staphylococcus maltophilia or a combination thereof.
[0338] [0338] Malaria is a disease transmitted by mosquitoes caused by Plasmodium parasites. The parasites are transmitted to people through the bites of infected female Anopheles mosquitoes. Five species of Plasmodium cause malaria in humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Among them, according to the World Health Organization (WHO), Plasmodium falciparum and Plasmodium vivax are responsible for the greatest threat. P. falciparun is the most prevalent malaria parasite on the African continent and is responsible for the majority of malaria-related deaths worldwide. P. vivax is the dominant malaria parasite in most countries outside the
[0339] [0339] As of 2015, 91 countries and areas have continued malaria transmission. According to the latest WHO estimates, there were 212 million cases of malaria in 2015 and 429,000 deaths. In areas with high malaria transmission, children under age 5 are particularly susceptible to infection, illness and death; more than two-thirds (70%) of all malaria deaths occur in this age group. Between 2010 and 2015, the under-5 mortality rate fell by 29% worldwide. However, malaria remains the leading cause of death for children under five, taking the life of a child every two minutes.
[0340] [0340] As described by the WHO, malaria is an acute febrile illness. In a non-immune individual, symptoms appear 7 days or more after the bite of the infected mosquito. The first symptoms - fever, headache, chills and vomiting - can be mild and difficult to recognize as malaria; however, if not treated within 24 hours, P. falciparum malaria can progress to serious illness, often leading to death.
[0341] [0341] Children with severe malaria often develop one or more of the following symptoms: severe anemia, respiratory distress in relation to metabolic acidosis, or cerebral malaria. In adults, multiple organ involvement is also common. In malaria-endemic areas, people can develop partial immunity, allowing asymptomatic infections to occur.
[0342] [0342] The development of rapid and efficient diagnostic tests is of high public health relevance. In fact, early diagnosis and treatment of malaria not only reduce disease and prevent deaths, but also contribute to reducing malaria transmission. According to WHO recommendations, all suspected malaria cases should be confirmed using parasite-based diagnostic tests (primarily using a rapid diagnostic test) before administering treatment (see "WHO Guidelines for the Treatment of Malaria". malaria", third edition, published in April 2015)
[0343] [0343] Resistance to antimalarial therapies represents a critical health problem that drastically reduces therapeutic strategies. In fact, as reported on the WHO website, the resistance of PP. falciparum to earlier generations of drugs such as chloroquine and sulfadoxine/pyrimethamine (SP) became widespread in the 1950s and 1960s, undermining malaria control efforts and reversing gains in child survival. Thus, the WHO recommends routine monitoring of antimalarial drug resistance. Indeed, accurate diagnosis can prevent inappropriate treatments and limit the extent of resistance to antimalarial drugs.
[0344] [0344] In this context, the WHO Global Technical Strategy for Malaria 2016-2030 - adopted by the World Health Assembly in May 2015 - provides a technical framework for all malaria-endemic countries. It is intended to guide and support regional and national programs as they work towards malaria control and elimination. The strategy sets ambitious but achievable global goals, including: . Reduce the incidence of malaria cases by at least 90% by 2030.
[0345] [0345] This strategy was the result of an extensive consultative process that lasted 2 years and involved the participation of over 400 technical experts from 70 Member States. It is based on 3 main axes: . guarantee universal access to prevention,
[0346] [0346] Treatment against Plasmodium includes aryl-amino alcohols such as quinine or quinine derivatives such as chloroquine, amodiaquine, mefloquine, piperaquine, lumefantrine, primaquine; lipophilic analogue of hydroxynaphthoquinone, such as atovaquone; antifolate drugs such as the sulfa drugs sulfadoxine, dapsone and pyrimethamine; proguanil; the atovaquone/proguanil combination; athemisin drugs; and combinations thereof.
[0347] [0347] Target sequences that are diagnostic for the presence of a mosquito-borne pathogen include sequences that are diagnostic for the presence of Plasmodium, especially Plasmodia species that affect humans such as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi, including their genome sequences.
[0348] [0348] Target sequences that are diagnostic for monitoring drug resistance to treatment against Plasmodium, mainly Plasmodia species that affect humans like Plasmodium falciparum, Plasmodium vivax,
[0349] [0349] Additional target sequences include sequences “include target molecules/nucleic acid molecules encoding proteins involved in essential biological processes for the Plasmodium parasite, and notably, carrier proteins such as drug/metabolite carrier family proteins, the cassette protein of ATP binding (ABC) involved in substrate translocation, such as ABC transporter subfamily C or Oo Na'/H' exchanger, membrane glutathione S-transferase; proteins involved in the folate pathway, such as dihydropteroate synthase, dihydrofolate reductase activity or dihydrofolate reductase thymidylate synthase; and proteins involved in the translocation of protons across the inner mitochondrial membrane and mainly the cytochrome b complex. Additional targets may also include the gene(s) encoding heme polymerase.
[0350] [0350] Additional target sequences include target molecules/nucleic acid molecules encoding proteins involved in essential biological processes that can be selected from the P. falciparum chloroquine resistance transporter gene (pfcrt), the multidrug resistance transporter P falciparum 1 (pfimdrl1), the multidrug resistance-associated protein gene
[0351] [0351] Several mutations, notably single-point mutations, have been identified in the proteins that are the targets of current treatments and associated with specific resistance phenotypes. Therefore, the invention allows for the detection of various resistance phenotypes of mosquito-borne parasites such as plasmodium.
[0352] [0352] The invention makes it possible to detect one or more mutations, especially one or more single nucleotide polymorphisms in target nucleic acids/molecules. Therefore, any of the mutations below, or their combination thereof, can be used as drug resistance markers and can be detected in accordance with the invention.
[0353] [0353] Single point mutations in P. falciparum K13 include the following single point mutations at positions 252, 441, 446, 449, 458, 493, 539, 543, 553, 561, 568, 574,
[0354] [0354] In P. falciparum dihydrofolate reductase (DHFR) (PEFDHFR-TS, PFDO830w), important polymorphisms include mutations at positions 108, 51, 59 and 164, notably 108D, 164L, 511 and 59R, which modulate the pyrimethamine resistance. Other polymorphisms also include 437G, 581G, 540E, 436A and 6138, which are associated with sulfadoxine resistance. Additional observed mutations include Serl108Asn, AsnSlIle, Cys59Arg, Ilel64Leu, Cys50Arg, Ilel64Leu, Asnl8S8Lys, Serl89Arg and Val213Ala, Serl08Thr and Alal6Val. Serl08Asn, AsnsSlIle, Cys59Arg, Ilel64Leu, Cys50Arg, TIlel64Leu mutations are notably associated with pyrimethamine-based therapy and/or resistances to chloroguanine-dapsone combination therapy. Cycloguanil resistance appears to be associated with the Serl08Thr and Alal6Val double mutations. Dhfr amplification may also be of high relevance for therapy resistance, notably pyrimethamine resistance.
[0355] [0355] In P. falciparum dihydropteroate synthase (DHPS) (PFDHPS, PFO8 0095), important polymorphisms include mutations at positions 436, 437, 581 and 613 Ser436Ala/Phe, Ala437Gly, Lys540Glu, Ala581Gly and Ala613Thr/Ser. Polymorphisms at position 581 and/or 613 have also been associated with resistance to sulfadoxine-pyrimethamine-based therapies.
[0356] [0356] In the P. falciparum chloroquine resistance transporter (PFCRT), the polymorphism at position 76, notably the Lys76Thr mutation, is associated with chloroquine resistance. Other polymorphisms include Cys72Ser, Met74TI1e, Asn75Glu, Ala220Ser, Gln271Glu, Asn326Ser, Ile356Thr and Arg37l1Ile that may be associated with chloroquine resistance. PIfCRT is also phosphorylated at residues S33, S41l and T4l16, which can regulate transport activity or protein specificity.
[0357] [0357] In the P. falciparum multidrug resistance transporter 1 (PEMDRI1) (PFE1150w), polymorphisms were identified at positions 86, 184, 1034, 1042, notably Asn86Tyr, Tyrl184-Phe, Ser1034Cys Asnl042Asp and Aspl246Tyr and were reported as to influence susceptibility to lumefantrine, artemisinin, quinine, meflocine, halofantrine and chloroquine. Furthermore,
[0358] [0358] In P. falciparum multidrug resistance-associated protein (PfMRP) (gene reference PFAO5S90w), polymorphisms at positions 191 and/or 437 such as Y191H and A437S have been identified and associated with chloroquine resistance phenotypes.
[0359] [0359] In the P. falciparum NA+/H+ exchanger (PINHE) (ref PF13 0019), increased repeat DNNND in ms4670 microsatellite may be a marker of quinine resistance.
[0360] [0360] Mutations that alter the ubiquinol binding site of the cytochrome b protein encoded by the cytochrome b gene (cytb, mal mito 3) are associated with atovaquone resistance. Mutations at positions 26, 268, 276, 133 and 280 and especially Tyr26Asn, Tyr268Ser, M1331 and G280D may be associated with atovaquone resistance.
[0361] [0361] For example, in P Vivax, mutations in PvMDR1, the homolog of Pf MDR1, have been associated with chloroquine resistance, primarily the polymorphism at position 976, such as the Y976F mutation.
[0362] [0362] The above mutations are defined in terms of protein sequences. However, one skilled in the art is able to determine corresponding mutations, including SNPS, to be identified as a target nucleic acid sequence.
[0363] [0363] Other identified drug resistance markers are known in the art, for example, as described in "Susceptibility of Plasmodium falciparum to antimalarial drugs (1996-2004)"; WHO; Artemisinin and artemisinin-based combination therapy resistance (April 2016 WHO/HTM/GMP/2016.5); “Drug-resistant malaria: molecular mechanisms and implications for public health” FEBS Lett. 2011 Jun 6;585 (11):1551-62. doi:10.1016/j3.febslet.2011.04.042. Epub 2011 Apr 23. Review. PubMed PMID: 21530510; the contents of which are incorporated herein by reference.
[0364] [0364] As for the polypeptides that can be detected according to the present invention, gene products of all genes mentioned herein can be used as targets. Accordingly, it is contemplated that such polypeptides may be used for identification, typing and/or detection of drug resistant species.
[0365] [0365] In certain exemplary embodiments, the systems, devices, and methods disclosed herein are directed to detecting the presence of one or more mosquito-borne parasites in a sample, such as a biological sample obtained from a subject.
[0366] [0366] Due to the rapid and sensitive diagnostic capabilities of the modalities disclosed herein, detection of parasite type, down to a single nucleotide difference, and the ability to be implanted as a POC device, the modalities disclosed herein can be used as therapeutic regimens. guide, such as selecting the appropriate course of treatment. The modalities disclosed in this document can also be used to screen environmental samples (mosquito population, etc.) for the presence and typing of the parasite. The modalities can also be modified to detect mosquito-borne parasites and other mosquito-borne pathogens simultaneously. In some cases, malaria and other mosquito-borne pathogens may initially show similar symptoms. Thus, the ability to quickly distinguish the type of infection can guide important “treatment decisions. Other mosquito-borne pathogens that can be detected in conjunction with malaria include dengue, West Nile virus, chikungunya, yellow fever, filariasis, Japanese encephalitis, Saint Louis encephalitis, western equine encephalitis, eastern equine encephalitis, Venezuelan equine encephalitis, encephalitis LaCrosse, and Zika.
[0367] [0367] In certain exemplary embodiments, the devices, systems, and methods disclosed herein may be used to distinguish various species of mosquito-borne parasites in a sample.
[0368] [0368] In certain example embodiments, species identification can be performed based on genes that are present in multiple copies in the genome, such as mitochondrial genes such as CYTB. In certain exemplary embodiments, species identification can be performed based on highly expressed and/or highly conserved genes such as GAPDH, Histone H2B, enolase, or LDH.
[0369] [0369] In certain example modalities, a method or diagnosis is designed to screen “mosquito-borne parasites through multiple phylogenetic and/or phenotypic levels at the same time. For example, the method or diagnosis may comprise the use of multiple CRISPR systems with different guide RNAs. A first set of guide RNAs can distinguish, for example, between Plasmodium falciparum or Plasmodium vivax. These general classes can be further subdivided. For example, guide RNAs can be designed and used in the method or diagnosis that distinguishes drug-resistant strains, in general or with respect to a specific drug or drug combination. A second set of guide RNAs can be designed to distinguish microbes at the species level. Thus, a matrix can be produced identifying all species or subspecies of mosquito-borne parasites, further divided according to drug resistance. The foregoing is for example purposes only. Other means of classifying other types of mosquito-borne parasites are also contemplated and would follow the general structure described above.
[0370] [0370] In certain exemplary embodiments, the devices, systems, and methods disclosed herein may be used to screen for genes of interest from mosquito-borne parasites, e.g., drug resistance genes. Guide RNAs can be designed to distinguish between known genes of interest. Samples, including clinical samples, can then be screened using the modalities disclosed herein to detect one or more such genes. The ability to screen for drug resistance in POC would be of tremendous benefit in selecting an appropriate treatment regimen. In certain exemplary embodiments, drug resistance genes are genes that encode proteins such as carrier proteins, such as proteins from the drug/metabolite transporter family, the ATP-binding cassette (ABC) protein involved in substrate translocation, such as Carrier subfamily C or the Na'/H' exchanger; proteins involved in the folate pathway, such as dihydropteroate synthase, dihydrofolate reductase activity or dihydrofolate reductase thymidylate synthase; and proteins involved in the translocation of protons across the inner mitochondrial membrane and mainly the cytochrome b complex.
[0371] [0371] In some embodiments, a CRISPR system, detection system, or methods of using the same, as described here, may be used to determine the course of a mosquito-borne parasite outbreak. The method may comprise detecting one or more target sequences from a plurality of samples from one or more subjects, wherein the target sequence is a sequence from a mosquito-borne parasite that spreads or causes outbreaks. Such a method may further comprise determining a transmission pattern of mosquito-borne parasites or a mechanism involved in a disease outbreak caused by a mosquito-borne parasite. Samples may be derived from one or more humans and/or derived from one or more mosquitoes.
[0372] [0372] The pattern of pathogen transmission may comprise continued new transmissions from the natural reservoir of the mosquito-borne parasite or other transmissions (eg, through mosquitoes) following a single transmission from the natural reservoir or a mixture of both. In one embodiment, the target sequence is preferably a sequence within the genome of the mosquito-borne parasite or fragments thereof. In one embodiment, the pattern of transmission of mosquito-borne parasites is the initial pattern of transmission of mosquito-borne parasites, that is, at the beginning of the outbreak of mosquito-borne parasites. Determining the pattern of transmission of mosquito-borne parasites early in the outbreak increases the likelihood of stopping the outbreak as early as possible, thus reducing the possibility of local and international spread.
[0373] [0373] Determining the transmission pattern of mosquito-borne parasites may comprise detecting a sequence of mosquito-borne parasites, in accordance with the methods described herein. Determining the pattern of pathogen transmission may further comprise detecting shared intra-host variations in the sequence of mosquito-borne parasites among subjects and determining whether the shared intra-host variations show temporal patterns. Patterns in observed intra-host and inter-host variation provide important information about transmission and epidemiology (Gire, et al., 2014).
[0374] [0374] In addition to other sample types disclosed herein, the sample may be derived from one or more mosquitoes, for example, the sample may comprise mosquito saliva. BIOMARKER DETECTION
[0375] [0375] In certain exemplary embodiments, the systems, devices, and methods disclosed herein may be used for detection of biomarkers. For example, the systems, devices and methods disclosed herein can be used for SNP detection and/or genotyping. The systems, devices and methods disclosed herein may also be used for the detection of any disease state or disorder characterized by aberrant gene expression. Aberrant gene expression includes aberration in the expressed gene, location of expression, and level of expression. Several transcripts or protein markers related to cardiovascular, immune and cancer diseases, among other diseases, can be detected. In certain exemplary embodiments, the modalities disclosed herein can be used for detection of cell-free DNA from diseases involving lysis, such as liver fibrosis and restrictive/obstructive lung disease. In certain example modalities, the modalities may be used for faster detection and more portable prenatal testing of cell-free DNA. The modalities disclosed in this document can be used to track panels of different SNPs associated with, among others, cardiovascular health, lipid/metabolic signatures, ethnicity identification, paternity matching, human ID (e.g., suspect matching to a database criminal record of SNP signatures). The modalities disclosed herein may also be used for detection of cell-free DNA of mutations related to and released from cancer tumors. The modalities disclosed in this document “can also be used for meat quality detection, for example providing rapid detection of different animal sources in a given meat product. Modalities disclosed herein may also be used for detection of GMOs or editing of DNA-related genes. As described elsewhere herein, closely related genotypes/alleles or biomarkers (eg, having only a single nucleotide difference in a given target sequence) can be distinguished by introducing a synthetic mismatch into the gRNA.
[0376] [0376] In one aspect, the invention relates to a method for detecting target nucleic acids in samples,
[0377] [0377] The sensitivity of the assays described herein is well suited for the detection of target nucleic acids in a wide variety of biological sample types, including sample types in which the target nucleic acid is diluted or for which the sample material is limited. Biomarker screening can be performed on various types of samples, including but not limited to saliva, urine, blood, feces, sputum, and cerebrospinal fluid.
[0378] [0378] In certain embodiments, the present invention provides steps to obtain a sample of biological fluid (e.g., urine, blood plasma or serum, sputum, cerebral spinal fluid) and extract the DNA. The mutant nucleotide sequence to be detected may be a fraction of a larger molecule or may be present initially as a distinct molecule.
[0379] [0379] In certain embodiments, DNA is isolated from the plasma/serum of a cancer patient. For comparison, DNA samples isolated from neoplastic tissue and a second sample can be isolated from non-neoplastic tissue from the same patient (control), eg lymphocytes. The non-neoplastic tissue may be of the same type as the neoplastic tissue or from a different organ source. In certain embodiments, blood samples are collected and the plasma is immediately separated from the blood cells by centrifugation. Serum can be filtered and stored frozen until DNA extraction.
[0380] [0380] In certain example modalities, target nucleic acids are detected directly from a raw or unprocessed sample sample, such as blood, serum, saliva, cerebrospinal fluid, sputum, or urine. In certain exemplary embodiments, the target nucleic acid is cell-free DNA.
[0381] [0381] In one embodiment, circulating cells (e.g., circulating tumor cells (CTC)) can be analyzed with the present invention. Isolation of circulating tumor cells (CTC) for use in any of the methods described herein can be accomplished. Exemplary technologies that achieve specific and sensitive detection and capture of circulating cells that can be used in the present invention have been described (Mostert B, et al., Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer. Cancer Treat Rev 2009;35:463-474; and Talasaz AH, et al., Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device. Proc Natl Acad Sci US A. 2009;106:3970- 3975). As few as a CTC can be found in the background of 105-106 peripheral blood mononuclear cells (Ross AA, et al., Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques. Blood. 1993,82:2605-2610). the platform
[0382] [0382] The present invention also provides isolation of CTCs with CTC-Chip technology. The CTC-Chip is a microfluidic-based CTC capture device where blood flows through a chamber containing thousands of microwells coated with anti-EpCAM antibodies to which the CTCs bind (Nagrath S, et al. circulating in cancer patients by microchip technology. Nature. 2007;450:1235-1239). The CTC-Chip provides a significant increase in CTC counts and purity compared to the CellSearcho system (Maheswaran S, et al. Detection of mutations in EGFR in circulating lung-cancer cells, N Engl J Med. 2008; 359: 366- 377), both platforms can be used for downstream molecular analyses.
[0383] [0383] In certain embodiments, cell-free chromatin fragments are isolated and analyzed in accordance with the present invention. Nucleosomes can be detected in the serum of healthy individuals ( Stroun et al., Annals of the New York Academy of Sciences 906: 161-168 (2000 )), as well as individuals affected by a disease state. Furthermore, the serum concentration of nucleosomes is considerably higher in patients suffering from benign and malignant diseases such as cancer and autoimmune diseases (Holdenrieder et al (2001) Int J Cancer 95, 1 14-120, Trejo-Becerril et al ( 2003) Int J Cancer 104, 663-668 ; Kuroi et al 1999 Breast Cancer 6, 361-364 ; Kuroi et al (2001 ) Int J Oncology 19, 143-148 ; Amoura et al (1997) Arth Rheum 40, 2217- 2225; Williams et al (2001) J Rheumatol 28,
[0384] [0384] Thus, in another embodiment, isolated chromatin fragments are derived from circulating chromatin, preferably circulating mono- and oligonucleosomes. Isolated chromatin fragments can be derived from a biological sample. The biological sample may be from a subject or patient in need thereof. The biological sample can be sera, plasma, lymph, blood,
[0385] [0385] In certain embodiments, the present invention may be used to detect cell-free DNA (cfDNA). Cell-free DNA in plasma or serum can be used as a non-invasive diagnostic tool. For example, cell-free fetal DNA has been studied and optimized to test for compatible RhD factors, sex determination for X-linked genetic disorders, testing for single-gene disorders, identification of preeclampsia. For example, sequencing the fetal cell fraction of cfDNA in maternal plasma is a reliable approach to detecting copy number changes associated with fetal chromosome aneuploidy. For another example, cIDNA isolated from cancer patients has been used to detect mutations in key genes relevant to treatment decisions.
[0386] [0386] In certain exemplary embodiments, the present disclosure provides detection of cfDNA directly from a patient sample. In another embodiment of another example, the present disclosure provides enriching cfDNA using the enrichment modalities disclosed above and prior to detection of the target cfDNA.
[0387] [0387] In one embodiment, exosomes may be analyzed with the present invention. Exosomes are small extracellular vesicles that have been shown to contain RNA. Isolation of exosomes by ultracentrifugation, filtration, chemical precipitation, size exclusion chromatography and microfluidics is known in the art. In one embodiment, exosomes are purified using an exosome biomarker. Isolation and purification of exosomes from biological samples can be carried out by any known methods (see, for example, WO2016172598A11).
[0388] [0388] In certain embodiments, the present invention can be used to detect the presence of single nucleotide polymorphisms (SNPs) in a biological sample. SNPs may be related to maternity testing (eg, sex determination, fetal defects). They may be related to a criminal investigation. In one embodiment, a suspect in a criminal investigation may be identified by the present invention. Not being bound by forensic evidence based on nucleic acid theory may require the most sensitive assay available to detect the genetic material of a suspect or victim, because the samples tested can be limiting.
[0389] [0389] In other embodiments, SNPs associated with a disease are encompassed by the present invention. Disease-associated SNPs are well known in the art and one skilled in the art can apply the methods of the present invention to design suitable guide RNAs (see, for example, www.ncbi.nlm.nih.gov/clinvar term=human% 5Borgn%$5D).
[0390] [0390] In one aspect, the invention relates to a method for genotyping, such as SNP genotyping comprising: dispensing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system in accordance with with the invention as described herein; incubating the sample or set of samples under conditions sufficient to allow binding of one or more guide RNASs to one or more target molecules; activating the CRISPR effector protein through binding of one or more guide RNAs to one or more target molecules, wherein activation of the CRISPR effector protein results in the modification of the RNA-based masking construct such that a detectable positive signal is generated; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates the presence of one or more target molecules characteristic of a specific genotype in the sample.
[0391] [0391] In certain embodiments, the detectable signal is compared to (e.g., by comparing signal strength) one or more standard signals, preferably a synthetic standard signal, as for example illustrated in one embodiment in Figure 60. In certain embodiments , the pattern is or matches a specific genotype. In certain embodiments, the pattern comprises a particular SNP or other (single) nucleotide variation. In certain embodiments, the pattern is a genotype pattern (amplified by PCR). In certain embodiments, the pattern is or comprises DNA. In certain embodiments, the pattern is or comprises RNA. In certain embodiments, the pattern is or comprises RNA that is transcribed from DNA. In certain embodiments, the pattern is or comprises DNA that is reverse transcribed from RNA. In certain embodiments, the detectable signal is compared to one or more standards, each of which corresponds to a known genotype, such as an SNP or other (unique) nucleotide variation. In certain embodiments, the detectable signal is compared to one or more standard signals and the comparison comprises statistical analysis, such as by parametric or non-parametric statistical analysis, such as one-way or two-way ANOVA, etc. In certain embodiments, the detectable signal is compared to one or more standard signals and when the detectable signal does not deviate (statistically) significantly from the standard, the genotype is determined to be the genotype corresponding to said standard.
[0392] [0392] In other embodiments, the present invention allows rapid genotyping for emergency pharmacogenomics.
[0393] [0393] In certain example modalities, the availability of genetic material to detect an SNP in a patient makes it possible to detect SNPs without amplification of a DNA or RNA sample. In the case of genotyping, the biological sample tested is easily obtained. In certain exemplary embodiments, the incubation time of the present invention may be reduced. The assay can be performed in a period of time necessary for an enzymatic reaction to occur. One skilled in the art can perform biochemical reactions in 5 minutes (eg 5 minutes binding). The present invention may use an automated DNA extraction device to obtain DNA from blood. The DNA can then be added to a reaction that generates a target molecule for the effector protein. Immediately after generation of the target molecule, the masking agent can be cut off and a signal detected. In exemplary embodiments, the present invention allows rapid diagnosis of POC to determine a genotype prior to administering a drug (e.g., blood thinner). In the case where an amplification step is used, all reactions take place in the same reaction in a one-step process. In preferred embodiments, the POC assay can be performed in less than one hour, preferably 10 minutes, 20 minutes, 30 minutes, 40 minutes or 50 minutes.
[0394] [0394] In certain embodiments, the systems, devices and methods disclosed herein can be used to detect the presence or level of expression of long non-coding RNAs (lncRNAs). The expression of certain lncRNAs is associated with disease status and/or drug resistance. In particular, certain incursions (eg tcons 00011252, tcons 00010506, tcons 00026344, tcons 00015940, tcons 00028298, tcons 00026380, tcons 00026521, tcons 00026521, tcons 00016127, nr 125939 nr 033834, tcons 00021026, tcons 00006579 NR 109890 and NR 026873) are associated with resistance to cancer treatment, such as resistance to one or more BRAF inhibitors (e.g. Vemurafenib, Dabrafenib, Sorafenib, GDC-0879, PLX-4720 and LGX818) in the treatment of melanoma ( for example, nodular melanoma, lentigo maligna, lentigo maligna melanoma, acral lentiginous melanoma, superficial spreading melanoma, mucosal melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma and soft tissue melanoma). Detection of IncRNAs using the various modalities described in this document can facilitate disease diagnosis and/or selection of treatment options.
[0395] [0395] In one embodiment, the present invention can guide DNA or RNA-targeted therapies (e.g., CRISPR, TALE, zinc finger proteins, RNAi), particularly in environments where rapid delivery of therapy is important to outcomes of the therapy. treatment.
[0396] [0396] Cancer cells suffer a loss of genetic material (DNA) when compared to normal cells. This deletion of genetic material by which almost all, if not all, cancers are called "loss of heterozygosity" (LOH). Loss of heterozygosity (LOH) is a serious chromosomal event that results in the loss of the entire gene and the surrounding chromosomal region. Loss of heterozygosity is a common occurrence in cancer, where it may indicate the absence of a functional tumor suppressor gene in the lost region. However, a loss can be silent because there is still a functional gene on the other chromosome of the chromosome pair. The remaining copy of the tumor suppressor gene can be inactivated by a point mutation, leading to the loss of a tumor suppressor gene. The loss of genetic material from cancer cells can result in the selective loss of one of two or more alleles of a gene vital for cell viability or cell growth at a specific locus on the chromosome.
[0397] [0397] An "LOH marker" is the DNA of a microsatellite locus, a deletion, alteration or amplification which, when compared to normal cells, is associated with cancer or other diseases. An LOH marker is often associated with the loss of a tumor suppressor gene or another gene, usually related to the tumor.
[0398] [0398] The term "microsatellites" refers to small repetitive DNA sequences that are widely distributed in the human genome. A microsatellite is a tract of tandem-repeated (i.e. adjacent) DNA motifs that range in length from two to five nucleotides and are typically repeated from 5 to 50 times. For example, the sequence TATATATATA (SEQ ID NO: 431) is a dinucleotide microsatellite and GTCGTCGTCGTCGTC (SEQ ID NO: 432) is a trinucleotide microsatellite (with A being Adenine, G Guanine, C Cytosine and T-Thymine). Somatic changes in the repeat length of such microsatellites have been shown to represent a characteristic of tumors. The chi RNAs can be designed to detect these microsatellites. Furthermore, the present invention can be used to detect changes in repeat length as well as amplifications and deletions based on quantitation of the detectable signal. Certain microsatellites are located in flanking or intronic regulatory regions of genes, or directly in gene codons. Microsatellite mutations in these cases can lead to phenotypic changes and diseases, especially in triple expansion diseases such as fragile X syndrome and Huntington's disease.
[0399] [0399] Frequent loss of heterozygosity (LOH) in specific chromosomal regions has been reported in many types of neoplasms. Allelic losses in specific chromosomal regions are the most common genetic alterations observed in a variety of neoplasms, therefore microsatellite analyzes have been applied to detect DNA from cancer cells in samples of body fluids such as sputum for lung cancer and urine for bladder cancer. (Rouleau et al. Nature 363, 515-521 (1993 ); and Latif, et al. Science 260, 1317-1320 (1993 )). Furthermore, it has been established that markedly increased concentrations of soluble DNA are present in the plasma of individuals with cancer and some other diseases, indicating that cell-free serum or plasma can be used to detect cancer DNA with microsatellite abnormalities. (Kamp et al. Science 264, 436-440 (1994); and Steck, et al. Nat Genet. 15(4), 356-362 (1997)). Two groups reported microsatellite changes in plasma or serum from a limited number of patients with small cell lung cancer or head and neck cancer. (Hahn, et al. Science 271, 350-353 (1996 ); and Miozzo, et al. Cancer Res. 56, 2285-2288 (1996 )). Detection of loss of heterozygosity in tumors and serum from melanoma patients has also been shown previously (see, for example, US patent number US6465177B1).
[0400] [0400] Thus, it is advantageous to detect LOH markers in a subject suffering from or at risk of cancer. The present invention can be used to detect LOH in tumor cells. In one embodiment, circulating tumor cells can be used as a biological sample. In preferred embodiments, cell-free DNA obtained from serum or plasma is used to non-invasively detect and/or monitor LOH. In other embodiments, the biological sample can be any sample described herein (eg, a urine sample for bladder cancer). Not being bound by theory, the present invention can be used to detect LOH markers with improved sensitivity compared to any previous method, thus providing for early detection of mutational events. In one embodiment, LOH is detected in biological fluids, where the presence of LOH is associated with the occurrence of cancer. The method and systems described in this document represent a significant advance over previous techniques such as PCR or tissue biopsy, providing a non-invasive, rapid and accurate method to detect LOH of specific cancer-associated alleles. Thus, the present invention provides methods and systems that can be used to screen high-risk populations and monitor high-risk patients undergoing chemoprevention, chemotherapy, immunotherapy, or other treatments.
[0401] [0401] As the method of the present invention only requires DNA extraction from bodily fluids such as blood, it can be performed at any time and repeatedly on a single patient. Blood can be collected and monitored for LOH before or after surgery; before, during and after treatment, such as chemotherapy, radiation therapy, gene therapy, or immunotherapy; or during follow-up examination after treatment for disease progression, stability, or recurrence. Not being bound by theory, the method of the present invention can also be used to detect the presence or subclinical recurrence of disease with a LOH marker specific to that patient, since LOH markers are specific to an individual patient's tumor. The method can also detect whether multiple metastases may be present using tumor-specific LOH markers.
[0402] [0402] Histone variants, DNA modifications and histone modifications indicative of cancer or cancer progression may be used in the present invention. For example, U.S. patent publication 20140206014 describes that cancer samples had elevated levels of nucleosome H2AZ, macroH2Al.1, 5-methylcytosine, P-H2AX (Serl39) compared to healthy subjects. The presence of cancer cells in an individual can generate a higher level of cell-free nucleosomes in the blood as a result of increased apoptosis of cancer cells. In one embodiment, an antibody directed against apoptosis-associated markers, such as H2B Ser 14(P), can be used to identify unique nucleosomes that have been released from apoptotic neoplastic cells. Thus, DNA resulting from tumor cells can be advantageously analyzed in accordance with the present invention with high sensitivity and precision.
[0403] [0403] In certain embodiments, the method and systems of the present invention may be used in prenatal screening. In certain embodiments, cell-free DNA is used in a prenatal screening method. In certain embodiments, DNA associated with single nucleosomes or oligonucleosomes can be detected with the present invention. In preferred embodiments, detection of DNA associated with single nucleosomes or oligonucleosomes is used for prenatal screening. In certain embodiments, cell-free chromatin fragments are used in a prenatal screening method.
[0404] [0404] Prenatal diagnosis or prenatal screening refers to the testing of diseases or conditions in a fetus or embryo before birth. The aim is to detect birth defects such as neural tube defects, Down syndrome, chromosomal abnormalities, genetic disorders and other conditions such as spina bifida, cleft palate, Tay Sachs disease, sickle cell anemia, thalassemia, cystic fibrosis, muscular dystrophy and of the fragile X. Screening can also be used for prenatal sexual discernment. Common testing procedures include amniocentesis, ultrasound including nuchal translucency ultrasound, serum marker testing, or genetic screening. In some cases, tests are given to determine whether the fetus will be aborted, although doctors and patients also find it helpful to diagnose high-risk pregnancies earlier so that delivery can be scheduled in a tertiary hospital where the baby can receive adequate care. .
[0405] [0405] It has been realized that there are fetal cells that are present in the mother's blood and that these cells present a potential source of fetal chromosomes for DNA-based prenatal diagnosis.
[0406] [0406] The H3 class of histones consists of four different types of proteins: the main types, H3.1 and H3.2; The replacement type, H3.3; and the testis-specific variant, H3t. Although H3.1 and H3.2 are closely related, differing only in Ser96, H3.1 differs from H3.3 in at least 5 amino acid positions. Furthermore, H3.1 is highly enriched in fetal liver, compared to its presence in adult tissues, including liver, kidney, and heart. In adult human tissue, the H3.3 variant is more abundant than the H3.1 variant, while the reverse is true for fetal liver. The present invention can use these differences to detect fetal nucleosomes and fetal nucleic acid in a maternal biological sample that comprises fetal and maternal cells and/or fetal nucleic acid.
[0407] [0407] In one embodiment, fetal nucleosomes may be obtained from blood. In other embodiments, fetal nucleosomes are obtained from a sample of cervical mucus. In certain embodiments, a sample of cervical mucus is obtained by swabbing or washing a pregnant woman early in the second trimester or late in the first trimester of pregnancy. The sample can be placed in an incubator to release the DNA trapped in the mucus. The incubator can be set to 37ºC. The sample can be shaken for approximately 15 to 30 minutes. The mucus can be further dissolved with a mukinase for the purpose of releasing DNA. The sample may also be subjected to conditions, such as chemical treatment and the like, as is well known in the art, to induce apoptosis to release fetal nucleosomes. Thus, a sample of cervical mucus can be treated with an agent that induces apoptosis, whereby fetal nucleosomes are released. With respect to enrichment of circulating fetal DNA, reference is made to U.S. patent publications 20070243549 and
[0408] [0408] Prenatal screening in accordance with the present invention may be for a disease that includes, but is not limited to, Trisomy 13, Trisomy 16, Trisomy 18, Klinefelter syndrome (47, XXY), (47, XYY) and (47, XXX), Turner Syndrome, Down Syndrome (Trisomy 21), Cystic Fibrosis, Huntington's Disease, Beta Thalassemia, Myotonic Dystrophy, Sickle Cell Anemia, Porphyria, Fragile X Syndrome, Robertsonian Translocation, Angelman Syndrome, DiGeorge syndrome and Wolf-
[0409] [0409] Various other aspects of the invention pertain to the diagnosis, prognosis and/or treatment of defects associated with a wide range of genetic diseases which are described in more detail on the website of the National Institutes of Health under the topic subsection Genetic Disorders (site at health .nih.gov/topic/Genetic Disorders).
[0410] [0410] In certain embodiments, the present invention may be used to detect cancer-associated genes and mutations. In certain embodiments, mutations associated with resistance are detected. Amplification of resistant tumor cells or emergence of resistant mutations in clonal populations of tumor cells may arise during treatment (see, for example, Burger JA, et al., Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition. Nat Commun. 2016 May 20;7:11589; Landau DA, et al., Mutations driving CLL and their evolution in progression and relapse. Nature. 2015 Oct 22;526(7574):525-30; Landau DA, et al. , Clonal evolution in hematological malignancies and therapeutic implications. Leukemia. 2014 Jan;28(1):34-43; and Landau DA, et al., Evolution and impact of subclonal mutations in chronic lymphocytic leukemia.
[0411] [0411] In certain modalities, mutations occur in individual cancers that can be used to detect cancer progression. In one embodiment, mutations related to the cytolytic activity of T cells against tumors have been characterized and can be detected by the present invention (see, for example, Rooney et al., Molecular and genetic properties of tumors associated with local immune cytolytic activity, Cell. 2015 January 15; 160(1-2): 48-61). Personalized therapies can be developed for a patient based on the detection of these mutations (see eg WOZ2016100975Al). In certain embodiments, cancer-specific mutations associated with cytolytic activity may be a mutation in a gene selected from the group consisting of CASP8, B2M, PIK3CA,
[0412] [0412] In certain embodiments, the present invention is used to detect a cancer mutation (eg, resistance mutation) during the course of a treatment and after the end of treatment. The sensitivity of the present invention can allow non-invasive detection of clonal mutations that arise during treatment and can be used to detect a recurrence in the disease.
[0413] [0413] In certain example modalities, detection of microRNAs (miRNA) and/or miRNA signatures of differentially expressed miRNA, can be used to detect or monitor the progression of a cancer and/or detect drug resistance for a therapy of cancer. As an example, Nadal et al. (Nature Scientific Reports, (2015) doi: 10.1038/srepl12464) describe mRNA signatures that can be used to detect non-small cell lung cancer (NSCLC).
[0414] [0414] In certain example modalities, the presence of resistance mutations in clonal subpopulations of cells can be used in determining a treatment regimen. In other modalities, therapies tailored to the treatment of a patient may be administered based on common tumor mutations. In certain modalities, common mutations arise in response to treatment and lead to drug resistance. In certain embodiments, the present invention can be used in monitoring patients for cells that acquire a mutation or amplification of cells that harbor such drug-resistant mutations.
[0415] [0415] Treatment with various chemotherapeutic agents, particularly with targeted therapies such as tyrosine kinase inhibitors, often leads to new mutations in target molecules that resist the activity of the therapy. Multiple strategies to overcome this resistance are being evaluated, including the development of second-generation therapies that are unaffected by these mutations and treatment with multiple agents, including those that act downstream of the resistance mutation. In one exemplary embodiment, a mutation common to ibrutinib, a molecule that targets Bruton's tyrosine kinase (BTK) and used for CLL and certain lymphomas, is a cysteine to serine change at position 481 (BTK/C481S). Erlotinib, which targets the tyrosine kinase domain of the Epidermal Growth Factor Receptor (EGFR), is commonly used in the treatment of lung cancer, and resistant tumors invariably develop after therapy. A common mutation found in resistant clones is a threonine to methionine mutation at position 790.
[0416] [0416] Non-silent mutations shared among cancer patient populations and common resistant mutations that can be detected with the present invention are known in the art (see, for example, WO/2016/187508). In certain modalities, drug resistance mutations can be induced by treatment with ibrutinib, erlotinib, imatinib, gefitinib, crizotinib, trastuzumab, vemurafenib, RAF/MEK, checkpoint blocking therapy, or antiestrogen therapy. In certain embodiments, cancer-specific mutations are present in one or more genes encoding a protein selected from the group consisting of Programmed Death Ligand 1 (PD-Ll), androgen receptor (AR), Bruton's Tyrosine Kinase (BTK) ), Epidermal Growth Factor Receptor (EGFR), BCR-Abl, c-kit, PIK3CA, HER2, EML4-ALK, KRAS, ALK, ROSl, AKT1l, BRAF, MEK1l, MEK2, NRAS, RAC1 and ESR1.
[0417] [0417] Immune checkpoints are inhibitory pathways that slow or stop immune reactions and prevent excessive tissue damage from uncontrolled immune cell activity. In certain embodiments, the targeted immune checkpoint is the programmed death-1 gene (PD-1 or CD279) (PDCD1). In other embodiments, the targeted immune checkpoint is cytotoxic T-lymphocyte-associated antigen (CTLA-4). In additional embodiments, the targeted immune checkpoint is another member of the CD28 and CTLA4 Ig superfamily, such as BTLA, LAG3, ICOS, PDL1, or KIR. In other additional embodiments, the targeted immune checkpoint is a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3.
[0418] [0418] Recently, gene expression in tumors and their microenvironments have been characterized at the cell level (see, for example, Tirosh, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single cell RNA-seq. Science 352, 189- 196, doi:10.1126/science.aad0501 (2016)); Tirosh et al., Single-cell RNA-segq supports a developmental hierarchy in human oligodendroglioma. Nature. 2016 Nov 10;539(7628):309-313. doi: 10.1038/nature20123. Epub 2016 Nov 2; and International patent publication WO 2017004153 A1). In certain embodiments, gene signatures can be detected using the present invention. In one embodiment, complement genes are monitored or detected in a tumor microenvironment. In one embodiment, the MITF and AXL programs are monitored or detected. In one embodiment, a tumor-specific stem cell or progenitor cell signature is detected. Such signatures indicate the status of an immune response and the status of a tumor. In certain embodiments, the status of a tumor in terms of proliferation, resistance to treatment, and abundance of immune cells can be detected.
[0419] [0419] Thus, in certain embodiments, the invention provides low-cost, rapid, multiplexed cancer detection panels for circulating DNA, such as tumor DNA, particularly to monitor disease recurrence or the development of common resistance mutations.
[0420] [0420] The modalities disclosed herein may also be useful in other immunotherapy contexts. For example, in some modalities, methods of diagnosing, prognosticating and/or staging an immune response in a subject comprise detecting a first level of expression, activity and/or function of one or more biomarkers and comparing the detected level with a level of control where a difference in the detected level and the control level indicates the presence of an immune response in the subject.
[0421] [0421] In certain embodiments, the present invention can be used to determine the dysfunction or activation of tumor-infiltrating lymphocytes (TIL). TILS can be isolated from a tumor using known methods. TILs can be analyzed to determine whether they should be used in adoptive cell transfer therapies. In addition, chimeric antigen receptor T cells (CAR T cells) can be analyzed for a signature of dysfunction or activation prior to administering them to a subject. Exemplary signatures for dysfunctional and activated T cells have been described (see, for example, Singer M, et al., A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells. Cell. 2016 Sep 8;166(6): 1500-1511.e9. doi: 10.1016/j.cell.2016.08.052).
[0422] [0422] In some embodiments, C2c2 is used to assess this status of immune cells such as T cells (eg CD8+ and/or CD4+ T cells). In particular, T cell activation and/or dysfunction can be determined, for example, on the basis of genes or gene signatures associated with one or more of the T cell states. In this way, c2c2 can be used to determine the presence of T cells. one or more T cell subpopulations.
[0423] [0423] In some embodiments, C2c2 may be used in a diagnostic assay or may be used as a method to determine whether a patient is suitable for administering an immunotherapy or other type of therapy. For example, detection of gene signatures or biomarkers can be performed via c2c2 to determine whether a patient is responding to a particular treatment or, if the patient is not responding, whether this may be due to T cell dysfunction. information about the types of therapy the patient is best suited to receive. For example, whether the patient should receive immunotherapy.
[0424] [0424] In some embodiments, the systems and assays disclosed herein may allow clinicians to identify whether a patient's response to a therapy (e.g., an adoptive cell transfer therapy (ACT)) is due to cellular dysfunction and, if any, levels of upregulation and upregulation in the biomarker signature will allow the issues to be resolved. For example, if a patient receiving ACT does not respond, cells administered as part of ACT can be analyzed by an assay disclosed herein to determine the relative level of expression of a biomarker signature known to be associated with cellular activation and/or states. of dysfunction. Whether a particular receptor or inhibitory molecule is upregulated in cells
[0425] [0425] In certain exemplary embodiments, the systems, methods, and devices described herein may be used to screen for gene signatures that identify a specific cell type, cell phenotype, or cell state. Likewise, through the use of methods such as packed detection, the modalities disclosed in this document can be used to detect transcriptomes. gene expression data is highly structured, so the expression level of some genes is predictive of the expression level of others. The knowledge that gene expression data are highly structured allows us to assume that the number of degrees of freedom in the system is small, which allows us to assume that the basis for calculating the relative abundance of genes is scarce. It is possible to make several biologically motivated assumptions that allow Applicants to retrieve nonlinear interaction terms during subsampling without having any specific knowledge of which genes are likely to interact. In particular, if Claimants assume that the genetic interactions are low-ranking, sparse, or a combination thereof, the actual number of degrees of freedom is small relative to the full combinatorial expansion, which allows Claimants to deduce the complete nonlinear landscape. with a relatively small number of disturbances.
[0426] [0426] Alternatively, the modalities described herein may be used to detect nucleic acid identifiers. Nucleic acid identifiers are non-coding nucleic acids that can be used to identify a specific article. Examples of nucleic acid identifiers, such as DNA watermarks, are described in Heider and Barnekow. “DNA watermarks: A proof of concept” BMC Molecular Biology 9:40 (2008). Nucleic acid identifiers can also be a nucleic acid barcode. A nucleic acid-based barcode is a short sequence of nucleotides (e.g., DNA, RNA, or combinations thereof) that is used as an identifier for an associated molecule, such as a target molecule and/or target nucleic acid. A nucleic acid barcode can be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, in length. 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 nucleotides and can be in single or double stranded form. One or more nucleic acid barcodes may be attached or "tagged" to a target molecule and/or target nucleic acid. This binding can be direct (e.g. covalent or non-covalent binding of the barcode to the target molecule) or indirect (e.g. via an additional molecule, e.g. a specific binding agent such as an antibody (or other protein). ) or a barcode receiving adapter (or other nucleic acid molecule). The target molecule and/or target nucleic acids can be tagged with multiple nucleic acid barcodes in a combinatorial manner, such as a nucleic acid bars. Typically, a nucleic acid bar code is used to identify target molecules and/or target nucleic acids as belonging to a specific compartment (e.g., a distinct volume), having a specific physical property (e.g., affinity, length, sequence, etc.), or having been subjected to certain treatment conditions. The target molecule and/or the target nucleic acid can be associated with various b codes nucleic acid arrays to provide information on all these resources (and more). Methods for generating nucleic acid barcodes are disclosed, for example, in International Patent Application Publication WO/2014/047561.
[0427] [0427] The application further provides C2c2 orthologs that demonstrate robust activity, making them particularly suitable for different RNA cleavage and detection applications. These orders include, but are not limited to, those described here. More particularly, an ortholog that has been shown to have stronger activity than the others tested is the C2c2 ortholog identified from the organism Leptotrichia wadei (LwC2c2). The application thus provides methods for modifying a target locus of interest, comprising delivering to said locus an unnatural or engineered composition comprising a C2c2 effector protein, more particularly a C2c2 effector protein with increased activity, as described herein, and one or more acids component nucleic acids, wherein at least the one or more nucleic acid components are engineered, the one or more nucleic acid components directs the complex to the target of interest, and the effector protein forms a complex with the one or more nucleic acid components and the complex binds to the target locus of interest. In particular embodiments, the target locus of interest comprises RNA. The application further provides for the use of the Cc2 effector proteins with increased activity in RNA sequence-specific interference, RNA sequence-specific gene regulation, tracking of RNA or RNA or lincRNA or non-coding RNA or nuclear RNA or mMRNA products, mutagenesis, in situ hybridization by fluorescence or reproduction.
[0428] [0428] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
[0429] [0429] There are two ways to perform a C2c2 diagnostic test for DNA and RNA. This protocol can also be used with protein detection variants after administration of detection aptamers. The first is a two-step reaction, where amplification and detection of C2c2 are done separately. The second is where everything is combined in a reaction and this is called a two-step reaction. It is important to keep in mind that amplification may not be necessary for higher concentration samples, so it is good to have a separate C2c2 protocol that does not have amplification built in.
[0430] [0430] CRISPR Effector Only - No Amplification: Table 7 Component Volume (UL) Protein (44 nM final) 2 CcrRNA (12 nM final) 1 background target (100 ng total) 1 target RNA (variable) 1 Probe with sensor of RNA (125 nM) 4 MgCl>7 (6 mM final) 2
[0431] [0431] The reaction buffer is: 40 mM Tris-HCl, 60 mM NaCl, pH 7.3
[0432] [0432] Carry out this reaction for 20 min-3 hours at 37°C. Read with excitation: 485 nm/20 nm, emission: 528 nm/20 nm. A signal for single molecule sensitivity can be detected as early as 20 minutes, but it is clear that the sensitivity is higher for longer reaction times.
[0434] [0434] The reaction buffer is: 40 mM Tris-HCl, 60 mM NaCl, pH 7.3
[0435] [0435] Perform for 20 min - 3 hours. The minimum detection time is about 20 minutes to see the sensitivity of a molecule. Carrying out the reaction longer only reinforces the sensitivity.
[0436] [0436] The NEB kit mentioned is the HighScribe T7 High Yield Kit. To resuspend the buffer, use a 1.5x concentration: resuspend three tubes of lyophilized substrate in 59 ul of buffer and use in the above mix. Each reaction is 20 uL, which is enough for 5 reactions. Single molecule sensitivity with this reaction was observed within 30 to 40 minutes.
[0437] [0437] Rapid, inexpensive, and sensitive nucleic acid detection can aid in point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-targeted and RNA-targeted CRISPR effector Casl3a (formerly known as C2c2) exhibits a "side effect" of promising RNAse activity in target recognition. We combine the Casl3a side effect with isothermal amplification to establish a CRISPR-based diagnosis (CRISPR-Dx), providing rapid detection of DNA or RNA with atomole sensitivity and single-base mismatch specificity. We use this Casl3a-based molecular detection platform called SHERLOCK (UNnLOCKing of High Sensitivity Specific Enzyme Reporter) to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. . In addition, SHERLOCK reaction reagents can be lyophilized for cold chain independence and long-term storage, and easily reconstituted on paper for field applications.
[0438] [0438] The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity on a portable platform can aid in disease diagnosis and monitoring, epidemiology and general laboratory tasks. Although there are methods to detect nucleic acids (1-6), they have trade-offs between sensitivity, specificity, simplicity, cost and speed. Microbial Clustered Regularly Interleaved Short Palindromic Repeats (CRISPR) and CRISPR-associated adaptive immune systems (CRISPR-Cas contain programmable endonucleases that can be harnessed for CRISPR-based diagnosis (CRISPR-Dx). , 8), RNA-guided effector RNases such as Casl3a (formerly known as C2c2) (8) can be reprogrammed with CRISPR RNAs (crRNAs) (9-11) to provide a platform for RNA-specific detection. its RNA target, activated Casl3a engages in the "collateral" cleavage of nearby untargeted RNAs (10).This crRNA-programmed collateral cleavage activity allows Casl3a to detect the presence of a specific RNA in vivo triggering programmed cell death (10) ) or in vitro by unspecific degradation of labeled RNA (10, 12). Here we describe SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), a platform for in vitro nucleic acid detection with atomole sensitivity based on nucleic acid amplification and 3 Caslo3a-mediated collateral cleavage of a commercial reporter RNA (12), allowing real-time detection of the target (Fig. 17).
[0439] [0439] For the in vivo bacterial efficiency assay, the C2c2 proteins of Leptotrichia wadei F0279 and Leptotrichia shahii were sorted as codon-optimized genes for mammalian expression (Genscript, Jiangsu, China) and cloned into pACYCI184 backbones, along with the corresponding forward repeats, which flanked a beta-targeting or non-targeting beta-lactamase spacer. Spacer expression was driven by a J23119 promoter.
[0440] [0440] For protein purification, mammalian codon-optimized C2c2 proteins were cloned into the bacterial expression vector for protein purification (6x His/Twin Strep SUMO, a pET-based expression vector received as a gift from Ilya Finkelstein).
[0441] [0441] In vivo efficiency plasmids LwC2c2 and LshCc2c2 and a previously described beta-lactamase plasmid (Abudayyeh 2016) were co-transformed into competent NovaBlue Singles (Millipore) cells at 90ng and ng, respectively. After transformation, cell dilutions were plated on the ampicillin and coramphicol LB-agar plate and incubated overnight at 37°C. Colonies were counted the next day.
[0442] [0442] Nucleic acid targets were PCR amplified with KAPA Hifi Hot Start (Kapa Biosystems), gel extracted and purified using the MinElute gel extraction kit (Qiagen). Purified dsDNA was incubated with T7 polymerase overnight at 30°C using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) and the RNA was purified with the MEGAClear Transcription Clean-up Kit (Thermo Fisher).
[0443] [0443] For the preparation of crRNA, the constructs were ordered as DNA (Integrated DNA Technologies) with a T7 promoter sequence attached. The crRNA DNA was annealed in a short T7 primer (final concentrations 10 uM) and incubated with T7 polymerase overnight at 37°C using the HiScribe T7 Quick High Yield RNA Synthesis Kit.
[0444] [0444] Details of the NASBA reaction are described in [Pardee 2016]. For a total reaction volume of 20 µl, 6.7 µl of reaction buffer (Life Sciences, NECB-24), 3.3 µl of Nucleotide Mix (Life Sciences, NECN-24), 0.5 µl of free water of nuclease, 0.4 µl of 12.5 µM NASBA primers, 0.1 µl of RNase inhibitor (Roche, 03335402001) and 4 µl of RNA amplicon (or water for the negative control) were mounted at 4°C and incubated at 65°C for 2 min and then at 41°C for 10 min. 5 µL of the enzyme mixture (Life Sciences, NEC-1-24) was added to each reaction, and the reaction mixture was incubated at 41°C for 2 hours. The NASBA primers used were 5'-AATTCTAATACGACTCACTATAGGGGGATCCTCTAGAAATATGGATT-3' (SEQ ID NO: 16) and 5'-CTCGTATGTTGTGTGGAATTGT-3' (SEQ ID NO: 17), and the underlined part indicates the T7 promoter sequence.
[0445] [0445] Primers for RPA were designed using the NCBI Primer blast (Ye et al., BMC Bioinformaics 13, 134 (2012) using standard parameters, with the exception of amplicon size (between 100 and 140 nt), primer (between 54C and 67C) and primer size (between 30 and 35 nt) The primers were then ordered as DNA (Integrated DNA Technologies).
[0446] [0446] The RPA and RTI-RPA reactions performed were as instructed by TwistAmpo Basic or TwistAmpO Basic RT (TwistDx), respectively, with the exception that 280 mM MgAc was added before the input model. Reactions were carried out with inlet lul for 2 hours at 37°C, unless otherwise noted.
[0447] [0447] C2c2 bacterial expression vectors were transformed into competent Rosetta" 2 (DE3) pLysS Singles (Millipore) cells. A 16 ml starter culture was grown in Terrific Broth 4 growth medium (12 g/L tryptone, 24 g/L yeast extract, 9.4 g/L K2HPO, 2.2 g/L KH2PO4, Sigma) (TB) to inoculate 4L of TB, which was incubated at 37C, 300 RPM until an OD600 of 0 , 6. At this time, protein expression was induced by supplementation with IPTG (Sigma) to a final concentration of 500uM, and the cells were cooled to 18°C for 16h for protein expression. The cells were then centrifuged at 5200 g, 15 min, 4 C. Cell pellet was collected and stored at -80°C for further purification.
[0448] [0448] All subsequent steps of protein purification are carried out at 4°C. The cell pellet was ground and resuspended in lysis buffer (20 mM Tris-Hcl, 500 mM NaCl, 1 mM DTIT, pH 8.0) supplemented with protease inhibitors (complete Ultra EDTA-free tablets), lysozyme and benzonase, followed by (Sonifier 450, Branson, Danbury, CT) with the following conditions: 100 amplitude for 1 second on and 2 seconds off with a total sonication time of 10 minutes.
[0449] [0449] Detection assays were performed with 45nM purified LwC2c2 substrate reporter, 22.5nM cCcrRNA, 125nM substrate reporter (Thermo Scientific RNAse Alert v2), 2puL murine RNase inhibitors, 100ng of total background RNA and amounts input nucleic acid target variables, unless otherwise indicated, in the buffer nuclease assay (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl 2 , pH 7.3). If the input was amplified, DNA including a T7 promoter from an RPA reaction, the above C2c2 reaction was modified to include 1mM ATP, 1mM GTP, ImM UTP, ImM CTP and 0.6puL T7 polymerase mixture (NEB). The reactions were allowed to continue for 1-3 hours at 37°C
[0450] [0450] The combination of one pot reaction, RPA-DNA amplification, conversion of T7 polymerase DNA to RNA, and C2c2 detection was performed by integrating the above reaction conditions with the RPA amplification mixture. Briefly, a 50 ul one-pot assay consisted of 0.48 uM forward primer, 0.48 uM reverse primer, 1x RPA rehydration buffer, varying amounts of DNA input, 45nM LwC2c2 recombinant protein, 22 crRNA .5 nM, background 250 ng total RNA, 200 nM substrate reporter (RNase alert v2), 4 ul RNase inhibitor, 2 mM ATP, 2 mM GTP, 2 mM UTP, 2 mM CTP, 1 µl of T7 polymerase mix, 5 mM MgCl 2 and 14 mM MgAc.
[0451] [0451] To compare SHERLOCK quantification with other established methods, qPCR was performed on a ssDNA 1 dilution series. A TagMan probe and primer set (sequences below) were designed against ssDNA 1 and synthesized with IDT. Assays were performed with the TaqMan Fast Advanced Advanced Master Mix (Thermo Fisher) and measured on a Roche LightCycler 480.
[0452] [0452] To compare SHERLOCK quantification with other established methods, we performed RPA on a ssDNA 1 dilution series. To quantify real-time DNA accumulation, we added lx SYBR Green II (Thermo Fisher) to the typical RPA reaction mix described above, which provides a fluorescent signal that correlates with the amount of nucleic acid. The reactions were allowed to continue for 1 hour at 37°C in a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 minutes.
[0453] [0453] The preparation and processing of the lentivirus was based on previously known methods. Briefly, 10 µg of pSB700 derivatives that include a Zika or Dengue RNA fragment, 7.5 pg of psPAX2 and 2.5 pg of pMD2.G were transfected into HEK293FT cells (Life Technologies, R7007) using the HeBS- CaCl2. 28 hours after medium change, DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 4 mM GlutaMAX (ThermoFisher Scientific), the supernatant was filtered using a 0.45 µm syringe filter. The ViralBind Lentivirus Purification Kit (Cell Biolabs, VPK-104) and Lenti-X Concentrator (Clontech, 631231) were used to purify and prepare lentiviruses from the supernatant. Viral concentration was quantified using the QuickTiter Lentivirus kit (Cell Biolabs, VPK-1112). Viral samples were added to 7% human serum (Sigma, H4522), heated at 95°C for 2 min, and used as input into the RPA.
[0454] [0454] Suspected Zika positive human serum or urine samples were inactivated with AVL buffer (Qiagen) and RNA isolation was achieved with the QIAamp viral RNA minikit (Qiagen). Isolated RNA was converted to cDNA by mixing random primers, dNTPs and RNA sample, followed by heat denaturation for 7 minutes at 70°C. The denatured RNA was then reverse transcribed with Superscript III (Invitrogen), incubating at 22-25ºC for 10 minutes, 50ºC for 45 minutes, 55ºC for 15 minutes and 80ºC for 10 minutes. The cDNA was then incubated for 20 minutes at 37°C with RNAse H (New England Biolabs)
[0455] [0455] 2mL of saliva was collected from volunteers who were prevented from consuming food or drink 30 minutes before collection. The samples were then processed using the QIAampoê DNA Blood Mini Kit (Qiagen) as recommended by the kit protocol. For boiled saliva samples, 400 µl of phosphate-buffered saline (Sigma) was added to 100 µl of volunteer saliva and centrifuged for 5 min at 1800 g. The supernatant was decanted and the pellet was resuspended in phosphate-buffered saline with 0.2% Triton X-100 (Sigma) before incubation at 95°C for 5 min. 1 pl of the sample was used as a direct input in the RPA reactions.
[0456] [0456] A glass fiber filter paper (Whatman, 1827-021) was autoclaved for 90 minutes (Consolidated Stills and Sterilizers, MKII) and blocked in 5% nuclease-free BSA (EMD Millipore, 126609-10GM) during the night. After rinsing the papers once with nuclease-free water (Life technologies, AM9932), they were incubated with 4% RNAsecureIM (Life technologies, AM7006) at 60°C for 20 minutes and rinsed three more times with the nuclease-free water. The treated papers were dried for 20 min at 80°C on a hot plate (Collection
[0457] [0457] For experiments involving CRE detection, bacterial cultures were grown in Lysogeny Broth (LB) to the intermediate phase, then sedimented and subjected to gDNA extraction and purification using the Qiagen DNeasy Blood and Tissue Kit, using the protocol manufacturer's label for Gram-negative or Gram-positive bacteria, as appropriate. gDNA was quantified by the Quant-It dsDNA assay on a Qubit fluorometer and its quality evaluated via the absorbance spectrum of 200-300 nm on a Nanodrop spectrophotometer.
[0458] [0458] For experiments that discriminate between E. coli and P. aeruginosa, bacterial cultures were grown to early stationary phase in Luria-
[0459] [0459] To confirm the concentration of the standard ssDNA 1 and ssRNA 1 dilutions used in Figure 1C, we performed digital droplet PCR (ddPCR). For DNA quantification, droplets were made using the ddPCR Supermix for Probes (dUTP-free) with PrimeTime aPCR probes/primer assays designed to target the sSSDNA 1 sequence. For RNA quantification, droplets were made using the RT- One-step ddPCR for probes with PrimeTime qaPCR probes/primer assays designed to target the SssRNA 1 sequence. Droplets were generated in both cases using the QX200 droplet generator (BioRad) and transferred to a PCR plate. Droplet-based amplification was performed in a thermocycler as described in the kit protocol, and nucleic acid concentrations were subsequently determined by measurement on a QX200 droplet reader.
[0460] [0460] To create patterns for accurate calling of genotypes from human samples, we designed primers around the SNP target to amplify -200 bp regions of human genomic DNA representing each of the two homozygous genotypes. The heterozygous pattern was then made by mixing the homozygous patterns in a 1:1 ratio. These standards were then diluted to genome-equivalent concentrations (-0.56 fg/uL) and used as input to SHERLOCK alongside real human samples.
[0461] [0461] To calculate the background subtracted fluorescence data, the initial fluorescence of the samples was subtracted to allow comparisons between different conditions. Fluorescence for background conditions (no input or no crRNA conditions) was subtracted from the samples to generate subtracted background fluorescence.
[0462] [0462] Guide ratios for SNP or strain discrimination were calculated by dividing each guide by the sum of the guide values, to adjust for the overall sample-to-sample variation. Ratios of crRNA to SNP or strain discrimination were calculated to adjust for the overall sample-to-sample variation as follows: . o (m + n)A; RIAA; ros E ERA AIHERA Bi where Ai and Bi refer to the SHERLOCK intensity values for the replication technique i of the crRNAs that detect the A allele or B allele, respectively, for a given “individual. As we normally have four technical repeats per crRNA, m and n are equal to 4 and The denominator is equivalent to the sum of all eight SHERLOCK crRNA intensity values for a given locus and SNP and individual. Because there are two crRNAs, the average ratio of crRNAs in each of an individual's crRNAs will always add up to two. Therefore, in the ideal case of homozygosity, the average ratio of crRNA to positive CcrRNA allele will be two and the average ratio of crRNA to negative crRNA allele will be zero. In the ideal case of heterozygosity, the average ratio of CrRNA to each of the two crRNAs will be one.
[0463] [0463] The protospacer flanking site
[0464] [0464] We purified recombinant Lwcasl3a protein from E. coli (Fig. 2D-E) and tested its ability to cleave a 173 nt SSsRNA with every possible nucleotide of a protospacer flanking site (PFS) (A, U, C or G) (Fig. 2F). Similar to LshCasl3a, LwCasl3a can cleave a target with PFS A, U, or C, with less SSRNA activity with a PFS G. Although we see weaker activity against SssRNA 1 with a G PFS, we still see robust detection for the two target sites with G PFS motifs (Table 3; rs601338 crRNA and Zika targeting crRNA 2). It is likely that the H PFS is not required in all circumstances and that in many cases strong cleavage or collateral activity can be achieved with a G PFS.
[0465] [0465] Recombinant polymerase amplification (RPA) is an isothermal amplification technique that consists of three essential enzymes: a recombinase, single-stranded DNA-binding proteins (SSBs), and a strand-displacement polymerase. RPA overcomes many technical difficulties present in other amplification strategies, particularly the polymerase chain reaction (PCR), by not requiring temperature regulation, as all enzymes operate at a constant temperature around 37ºC. RPA replaces temperature cycling by global double-stranded template melting and primer annealing with an enzymatic approach inspired by in vivo DNA replication and repair. Recombinant-primary complexes scan double-stranded DNA and facilitate the exchange of strands at complementary sites. Tape switching is stabilized by SSBs, allowing the initiator to stay on. Spontaneous disassembly of the recombinase occurs in its ADP-bound state, allowing a strand-shift polymerase to invade and extend the primer, allowing amplification without complex instrumentation unavailable in point-of-care and field settings. Cyclic repetition of this process in a tempered range of 37-42°C results in exponential amplification of DNA. The original published formulation uses Bacillus subtilis Pol 1 (Bsu) as the strand-shifting polymerase, T4 uvsX as the recombinase, and T4 gp32 as the single-stranded DNA binding protein (2), although it is unclear which components are present in the formulation. current sold by TwistDx used in this study.
[0466] [0466] In addition, RPA may have several limitations: 1) Although the detection of Casl3a is quantitative (Fig. 15), real-time quantification of RPA can be difficult due to its rapid saturation when the recombinase uses up all the ATP available. While real-time PCR is quantitative due to the ability to switch amplification, RPA lacks a mechanism to tightly control the amplification rate. Some adjustments can be made to reduce the amplification rate, such as reducing available concentrations of magnesium or primers, lowering the reaction temperature, or designing inefficient primers. Although we see some instances of quantitative SHERLOCK, as in Fig. 31, 32 and 52, this is not always the case and depends on the model.
[0467] [0467] The modularity of SHERLOCK allows any amplification technique, even non-isothermal approaches, to be used prior to T7 transcription and Casl3a detection. This modularity is made possible by the compatibility of the T7 and Casl3a steps in a single reaction, allowing detection to be performed on any amplified DNA input that has a T7 promoter. Prior to using RPA, nucleic acid sequence based amplification (NASBA) (3, 4) was attempted for our detection assay (Fig. 10). However, NASBA did not dramatically improve the sensitivity of Casl3a (Figs. 11 and 53). Other amplification techniques that could be employed prior to detection include PCR, loop-mediated isothermal amplification (LAMP) (5), strand shift amplification (SDA) (6), helicase-dependent amplification (HDA) (7) and amplification test (NEAR) (8). The ability to swap any isothermal technique allows SHERLOCK to overcome the specific limitations of any amplification technique.
[0468] [0468] Previously, we showed that cleavage of the LshCasl3a target was reduced when there were two or more mismatches in the target:crRNA duplex, but was not affected by single mismatches, an observation we confirmed for collateral cleavage of LwCasl3a (Fig. 36A). We hypothesized that introducing an additional mutation into the CrRNA spacer sequence would destabilize collateral cleavage against a target with an additional mismatch (two mismatches in total), while maintaining collateral cleavage in the target as there would only be one mismatch.
[0469] [0469] For incompatibility detection of ZIKV and DENV strains, our full-length crRNA contained two mismatches (Fig. 37A, B). Due to the high sequence divergence between the strains, we were unable to find a continuous stretch of 28 nt with only a single nucleotide difference between the two genomes. However, we predicted that shorter crRNAs would still work and designed shorter 23nt crRNAs against targets in the two ZIKV strains that included a synthetic spacer sequence mismatch and only a target sequence mismatch. These crRNAs could still distinguish African and American strains of ZIKV (Fig. 36C). Subsequent tests of 23 nt and 20 nt crRNA show that reductions in spacer length reduce activity but maintain or increase the ability to discriminate single mismatches (Fig. 57A-G). To understand -. To better understand how synthetic mismatches can be introduced to facilitate discrimination of single nucleotide mutations, we grouped the synthetic mismatch into the first seven spacer positions at three different spacer lengths: 28, 23, and 20 nt (Fig. 57A). In a target with a mutation in the third position, Lwcasl3a shows maximum specificity when the synthetic mismatch is at the spacer position, with enhanced specificity at shorter lengths, albeit with lower levels of activity on the target (Fig. 57B-G). We also shifted the target mutation by positions 3-6 and found synthetic differences side by side in the spacer around the mutation (Fig. 58).
[0470] [0470] The evaluation of synthetic patterns created from the PCR amplification of the SNP loci allows the precise identification of the genotypes (Fig. 60A, B). By calculating all comparisons (ANOVA) between the SHERLOCK results of an individual sample and the synthetic patterns, each individual's genotype can be identified by finding the synthetic pattern that has the most similar SHERLOCK detection intensity (Fig. 60C, Fig. 60C, D). This SHERLOCK genotyping approach is generalizable to any SNP locus (Fig. 60E).
[0471] [0471] For the SHERLOCK cost analysis, reagents considered to be of negligible cost were omitted, including DNA templates for crRNA synthesis, primers used in RPA, common buffers (MgCl2, Tris HCl, glycerol, NaCl, DTT), glass microfiber filter paper and RNAsecure reagent. For DNA models, IDT's ultramer synthesis provides material for 40 in vitro transcription reactions (each being sufficient to -
[0472] [0472] In addition, for all experiments except paper assays, 384-well plates (Corning 3544) were used, at a cost of US$0.036/reaction. Because of the negligible cost, this was not included in the overall cost analysis. In addition, the SHERLOCK-POC does not require the use of a plastic container as it can easily be run on paper. The reading method for SHERLOCK used here was a plate reader equipped with a filter set or a monochromator. As a capital investment, the cost of the reader was not included in the calculation, as the cost drops precipitously as more reactions are performed on the instrument and is negligible. For POC applications, cheaper and more portable alternatives can be used, such as handheld spectrophotometers (9) or handheld electronic readers (4), which reduce the cost of instrumentation to <US200. While these more portable solutions reduce speed and readability compared to bulkier instruments, they allow for wider use.
[0473] [0473] The assay and systems described herein may generally comprise a two-step process of amplification and detection. During the first step, the nucleic acid sample, RNA or DNA, is amplified, for example by isothermal amplification. During the second step, the amplified DNA is transcribed into RNA and subsequently incubated with a CRISPR effector such as C2c2 and a crRNA programmed to detect the presence of the target nucleic acid sequence. To allow detection, a reporter RNA that has been labeled with a quenched fluorophore is added to the reaction. Collateral cleavage of the reporter RNA results in deactivation of the fluorophore and allows real-time detection of the nucleic acid target (Fig. 17A).
[0474] [0474] To obtain robust signal detection, a C2c2 ortholog was identified from the organism Leptotrichia wadei (LwC2cC2) and evaluated. The activity of the LwCc2c2 protein was evaluated by expressing it together with a synthetic CRISPR array in E. coli and programming it to cleave a target site on the beta-lactamase mRNA, which leads to the death of bacteria under ampicillin selection (Fig. 2B). ) Fewer surviving E. coli colonies were observed at the Lwc2c2 locus than at the LshC2c2 locus, demonstrating a higher cleavage activity of the LwC2c2 ortholog (Fig. 2C). The human codon-optimized LwC2c2 protein was then purified from E. coli (Fig. 2D-E) and its ability to cleave a 173 nt ssRNA analyzed with nucleotides from different protospacer flanking sites (PFS) (Fig. 2F). ). LwCc2c2 was able to separate each of the four possible PFS targets, with slightly less SSRNA activity with a G PFS.
[0475] [0475] Real-time measurement of LwC2c2 RNase collateral activity was measured using a commercially available RNA fluorescent plate reader (Fig. 17A). To determine the sensitivity of LwCc2c2 activity, LwCc2c2 was incubated with ssRNA target 1 (ssRNA 1) and a crRNA complementary to a site within the SSRNA target, along with the RNA sensor probe (Fig. 18). This produced a sensitivity of -50 µM (Fig. 27A), which, while more sensitive than other recent nucleic acid detection technologies (Pardee et al., 2014), is not sensitive enough for many diagnostic applications that require subfemtomolar detection performance (Barletta et al., 2004; Emmadi et al., 2011; Rissin et al., 2010; Song et al., 2013).
[0476] [0476] To increase sensitivity, an isothermal amplification step was added prior to incubation with Lwc2c2. Coupling LwC2c2-mediated detection with previously used isothermal amplification approaches, such as nucleic acid sequence-based amplification (NASBA) (Compton, 1991; Pardee et al., 2016), has improved sensitivity to some extent (Fig. 11). ). An alternative isothermal amplification approach, recombinase polymerase amplification (RPA) (Piepenbhurg et al., 2006), which can be used to exponentially amplify DNA in less than two hours, was tested. By adding a T7 RNA polymerase promoter to the RPA primers, the amplified DNA can be converted to RNA for subsequent detection by LwC2c2 (Fig. 17). Thus, in certain exemplary embodiments, the assay comprises the combination of RPA amplification, conversion of T7 RNA polymerase from DNA to RNA, and subsequent detection of the RNA by C2c2 de-blocking the fluorescence of an quenched reporter.
[0477] [0477] Using the example method on a synthesized DNA version of SsSsRNA 1, it was possible to achieve atomole sensitivity in the range of 1 to 10 molecules per reaction (Fig. 27B, left). In order to verify detection accuracy, the input DNA concentration was qualified with digital droplet PCR and confirmed that the lowest detectable target concentration (2 aM) was at the concentration of a single molecule per microliter. With the addition of a reverse transcription step, RPA can also amplify RNA in the form of dsDNA, allowing atomole sensitivity in ssRNA 1 to be achieved (27B, right). Likewise, the concentrations of the RNA targets were confirmed by digital droplet PCR. To assess the feasibility of the example method to function as a POC diagnostic test, the ability of all components - RPA, T7 polymerase amplification and Lwc2c2 detection - to work in a single reaction was tested and found atomole sensitivity with one version of an assay pot (Fig. 22).
[0478] [0478] It was then determined whether the test would be effective in infectious disease applications that require high sensitivity and could benefit from portable diagnosis. To test detection in a model system, lentiviruses were produced containing RNA fragments of the Zika virus genome and the related Dengue flavivirus (Dejnirattisai et al., 2016) and the number of viral particles quantified (Fig. 31A). Simulated virus levels were detected up to 2 am.
[0479] [0479] Zika viral RNA levels in humans have been reported to be as low as 3 x 10th copies/mL (4.9 £M) in patient saliva and 7.2 x 10th copies/mL (1.2 £M) M) in the patient's serum (Barzon et al., 2016; Gourinat et al., 2015; Lanciotti et al., 2008). In the patient samples obtained, concentrations as low as 1.25 x 10º copies/mL (2.1 aM) were observed. To assess whether the assay is able to detect Zika virus from low titer clinical isolates, viral RNA was extracted from the patients and reverse transcribed, and the resulting cDNA was used as input to the assay (Fig. 32A). Significant detection for human serum samples of Zika was observed at concentrations below 1.25 copy/ul (2.1 aM) (Fig. 32B). In addition, the signal from patient samples was predictive of Zika viral RNA copy number and could be used to predict viral load (Fig. 31F). To test for broad applicability to disease situations in which nucleic acid purification is not available, the detection of spiked SssRNA 1 in human serum was tested and the assay was determined to activate at serum levels of less than 2% (Fig. 33G). ).
[0480] [0480] To determine whether the assay could be used to distinguish bacterial pathogens, the 168 V3 region was selected as an initial target as the conserved flanking regions allow universal RPA primers to be used between bacterial species and the internal variable region , allowing the differentiation of species. A panel of 5 possible targeted crRNAs was designed for pathogenic strains and gDNA isolated from E. coli and Pseudomonas aeruginosa (Fig. 34A). The assay was able to distinguish gDNA from E. coli or P. aeruginosa and showed low background signal for crRNAs from other species (Fig. 34 A, B).
[0481] [0481] The assay can also be adapted to rapidly detect and distinguish bacterial genes of interest, such as antibiotic resistance genes. Carbapenem-resistant Enterobacteriaceae (CRE) are an important emerging public health challenge (Gupta et al., 2011). The assay's ability to detect carbapenem resistance genes was evaluated and whether the test could distinguish between different carbapenem resistance genes. Klebsiella pneumonia was obtained from clinical isolates containing Klebsiella pneumoniae carbapenemase (KPC) resistance genes or Klebsiella pneumoniae carbapenemase (KPC) resistance genes.
[0482] [0482] Certain CRISPR RNA-guided RNase orthologs, such as LshC2c2, have been shown not to readily distinguish single-base mismatches (Abudayyeh et al., 2016). As demonstrated here, LwC2c2 also shares this feature (Fig. 37A). To increase the specificity of LwCc2c2 cleavage, a system for introducing synthetic mismatches into the crRNA:target duplex was developed that increases overall mismatch sensitivity and allows for single-base mismatch sensitivity. Several crRNAs for target 1 were designed and included mismatches along the length of the crRNA (Fig. 37A) to optimize target cleavage and minimize cleavage of a target that differs by a single mismatch. These mismatches did not reduce the cleavage efficiency of SSRNA target 1, but significantly decreased the signal for a target that included an additional mismatch (SSRNA target 2).
[0483] [0483] Having demonstrated that C2c2 can be engineered to recognize single-base mismatches, it was determined whether this engineered specificity could be used to distinguish between closely related viral pathogens. Several crRNAs were designed to detect African or American Zika virus strains (Fig. 37A) and Dengue virus strains 1 or 3 (Fig. 37C). These CrRNAs included a synthetic mismatch in the spacer sequence, causing a single bubble to form when duplexed to the target deformation due to the synthetic mismatch. However, when the synthetic mismatch spacer is duplexed for off-target strain, two bubbles form due to synthetic mismatch and SNP mismatch. Synthetic mismatch crRNAs detected their corresponding strains with significantly higher signal than the off-target strain, allowing robust strain distinction (Fig. 37B, 37D) Due to the significant sequence similarity between the strains, it was not possible to find a stretch 28 nt continuum with only a single nucleotide difference between the two genomes in order to demonstrate a true single nucleotide strain distinction. However, it was predicted that the shorter cCcrRNAs would still function, as they do with LshC2c2 (Abudayyeh et al., 2016), and therefore the shorter 23 nt crRNAs were designed against targets in the two Zika strains that included a mismatch. synthetic in the spacer sequence and only one mismatch in the target sequence. These crRNAs were still able to distinguish African and American strains of Zika with high sensitivity (Fig. 36C).
[0484] [0484] Rapid genotyping of human saliva may be useful in emergency pharmacogenomic situations or in home diagnostics. To demonstrate the potential of the disclosed modalities for genotyping, five loci were chosen to compare C2c2 detection using 23andMe genotyping data as the gold standard (Eriksson et al., 2010) (Fig. 38A). The five loci encompass a wide range of functional associations, including sensitivity to drugs such as statins or acetaminophen, susceptibility to norovirus, and risk of heart disease (Table 12). Table 12: SNP variants tested pp eee Jestegoia — Saturated fat consumption and rs5082 APOA2Z weight gain rs1467558|CD44 Acetaminophen metabolism close to rs2952768|CREB1 4.5x morphine dependence increases risk of myopathy for rs4363657|SLCO1B1 statin users
[0485] [0485] Saliva from four human subjects was collected and genomic DNA purified using a simple commercial kit in less than one hour. The four subjects had a diverse set of genotypes at the five loci, providing a large enough sample space to compare the assay for genotyping. For each of the five SNP loci, an individual's genomic DNA was amplified using RPA with the appropriate primers, followed by detection with Lwc2c2 and pairs of crRNAs designed to specifically detect one of two possible alleles (Fig. 38B). The assay was specific enough to distinguish alleles with high significance and infer homozygous and heterozygous genotypes. As a DNA extraction protocol was performed on saliva prior to detection, the assay was tested to determine if it could be even more affordable for POC genotyping using saliva heated to 95°C for 5 minutes without any further extraction. The assay was able to correctly genotype two patients whose saliva was subjected only to heating for 5 minutes and then subsequent amplification and detection of C2c2 (Fig. 40B).
[0486] [0486] As the test is highly specific for single nucleotide differences in targets, a test was developed to determine if the test was sensitive enough to detect cancer mutations in cell-free DNA (cfDNA). cfDNA fragments are a small percentage (0.1% to 5%) of wild-type cfDNA fragments (Bettegowda et al., 2014; Newman et al., 2014; Olmedillas Lopez et al., 2016; Qin et al., 2016; Qin et al. ., 2016). A significant challenge in the field of cIfDNA is detecting these mutations because they are typically difficult to discover given the high levels of unmutated DNA found in background blood (Bettegowda et al., 2014; Newman et al., 2014; Qin et al., 2014; Qin et al. ., 2016). A POC cfDNA cancer test would also be useful for regular screening for the presence of cancer, especially for patients at risk of remission.
[0487] [0487] The ability of the assay to detect mutant DNA in wild-type background was determined by diluting target 1 of dsDNA into a background of ssDNA1 with a single mutation at the crRNA target site (Fig. 41A-B). Lwc2c2 was able to detect dsDNA 1 at levels as low as 0.1% of background dsDNA and within atmolar concentrations of dsDNA 1. This result shows that cleavage by LwC2c2 of the background mutant dsDNA 1 is low enough to allow robust detection of dsDNA on target in the 0.1% allelic fraction. At levels below 0.1%, background activity is likely to be an issue, preventing any further significant detection of the correct target.
[0488] [0488] As the assay could detect synthetic targets with allelic fractions in a clinically relevant range, it was evaluated whether the assay was capable of detecting cancer mutations in cfDNA. RPA primers for two different cancer mutations, EGFR L858R and BRAF V600E, were designed and commercial cfDNA standards were used with 5%, 1%, and 0.1% allelic fractions that resemble real human ciDNA samples for test. Using a pair of crRNAs that could distinguish the mutant allele from the wild-type allele (FIG. 38C), detection of the 0.1% allelic fraction was achieved for both mutant loci (FIG. 39 AB).
[0489] [0489] By combining the natural properties of C2c2 with isothermal amplification and a quenched fluorescent probe, the assay and systems disclosed herein have been demonstrated to be a versatile and robust method for detecting RNA and DNA and suitable for a variety of rapid diagnostics, including of infectious diseases and rapid genotyping. A major advantage of the assays and systems disclosed here is that a new POC test can be re-engineered and synthesized in a matter of days for as low as $0.6/test.
[0490] [0490] As many human disease applications require the ability to detect single mismatches, a rational approach was developed to design CcrRNAsS to be highly specific to a single mismatch in the target sequence by introducing a synthetic mismatch into the CrRNA spacer sequence. Other approaches to achieving specificity with CRISPR effectors rely on methods based on screening in dozens of guide projects (Chavez et al., 2016). Using engineered mismatch CcrRNAs, we demonstrated discrimination of Zika and dengue viral strains at sites that differ by a single mismatch, rapid genotyping of gDNA SNPs from human saliva, and detection of cancer mutations in cfiDNA samples.
[0491] [0491] The low cost and adaptability of the assay platform lends itself to other applications, including (i) general expertise in RNA/DNA quantification as a replacement for specific qPCR assays such as Taqgman, (ii) rapid and multiplexed detection of microarray-like RNA expression; and (iii) other sensitive detection applications, such as the detection of nucleic acid contamination from other sources in foods. Furthermore, C2c2 could potentially be used for the detection of transcripts in biological environments, such as in cells, and given the highly specific nature of C2c2 detection, it may be possible to trace the allelic-specific expression of transcripts or disease-associated mutations in cells. cheers. With the wide availability of aptamers, it may also be possible to detect proteins by coupling the detection of proteins by an aptamer to the revelation of a cryptographic amplification site for RPA followed by the detection of C2c2.
[0492] [0492] For robust signal detection, we identified a Casl3a ortholog from Leptotrichia wadei (Lwcasl3a), which exhibits higher RNA-guided RNase activity relative to Leptotrichia shahii Casl3a (LshCasl3a) (10) (Fig. 2, see also above "Characterization of cleavage requirements of LwCcasl3a ”“”). LwCasl3a incubated with ssRNA target 1 (SSRNA 1), CcrRNA and reporter (fluorescent quenched RNA) (Fig. 18) (13) produced a detection sensitivity of -50 £fM (Fig. 51, 15), which is not sensitive enough for many diagnostic applications (12, 14-16), so we explored the combination of Casl3a-based detection with different isothermal amplification steps (Fig. 10, 11, 53, 16) (17, 18) Of the methods explored, recombinase polymerase (RPA) amplification (18) provided the highest sensitivity and could be coupled to T7 transcription to convert amplified DNA to RNA for subsequent detection by LwCasl3a ( see also above "Discussion of amplification by rec mbinase polymerase (RPA) and other isothermal amplification strategies.”). We refer to this combination of RPA amplification, transcription of T7 RNA polymerase from amplified DNA to RNA, and detection of target RNA by collateral release mediated by collateral RNA cleavage of Casl3a of the reporter signal as SHERLOCK.
[0493] [0493] First, we determined the sensitivity of SHERLOCK for detecting RNA (when coupled with reverse transcription) or DNA targets. We achieved single-molecule sensitivity for RNA and DNA as verified by digital droplet PCR (ddPCR) (Fig. 27 51, 54A, B). Atomole sensitivity was maintained when we combined all SHERLOCK components in a single reaction, demonstrating the feasibility of this platform as a point-of-care (POC) diagnostic (Fig. 54C). SHERLOCK has similar sensitivity levels to ddPCR and quantitative PCR (qPCR), two established sensitive nucleic acid detection approaches, while RPA alone was not sensitive enough to detect low levels of target (Fig. 55A-D) . In addition, SHERLOCK shows less variation than ddPCR, qPCR, and RPA, as measured by the coefficient of variation between replicates (Fig. 55E-F).
[0494] [0494] Next, we examined whether SHERLOCK would be effective in infectious disease applications that require high sensitivity. We produced lentiviruses containing genome fragments of the Zika virus (ZIKV) or flavivirus-related dengue (DENV) (19) (Fig. 31A). SHERLOCK detected viral particles up to 2 aM and was able to discriminate between ZIKV and DENV (Fig. 31B). To explore the potential use of SHERLOCK in the field, we first demonstrated that lyophilized and subsequently rehydrated Casl3acrRNA complexes (20) could detect 20 fM of unamplified ssRNA 1 (Fig. 33A) and that target detection was also possible on paper. fiberglass (fig. 33B). The other components of SHERLOCK are also amenable to lyophilization: RPA is supplied as a lyophilized reagent at room temperature,
[0495] [0495] To increase the specificity of SHERLOCK, we introduced synthetic mismatches in the CrRNA:target duplex which allows Lwcasl3a to discriminate between targets that differ by a single base mismatch (Fig. 36A, B; see also above "Engineered Mismatches Design") . We designed several CrRNAs with synthetic mismatches in the spacer sequences to detect African or American strains of ZIKV (Fig. 37A) and strains 1 or 3 of DENV (Fig. 37C). Synthetic mismatch CrRNAs detected their corresponding strains with significantly higher signal (two-tailed Student's t test; p < 0.01) than the off-target strain, allowing robust strain discrimination based on single mismatches (Fig. 37B). , D, 36C). Further characterization revealed that the detection of Casl3a achieves maximum specificity while maintaining sensitivity on the target when a mutation is at position 3 of the spacer and the synthetic mismatch is at position 5 (Figs. 57 and 58). The ability to detect single-base differences opens up the opportunity to use SHERLOCK for rapid human genotyping.
[0496] [0496] The SHERLOCK platform lends itself to other applications, including (i) general RNA/DNA quantification rather than specific qPCR assays such as TaqgMan, (ii rapid multiplexed detection of RNA expression, and (iii) other applications of sensitive detection, such as detection of nucleic acid contamination. In addition, Casl3a could detect transcripts in biological environments and track the allele-specific expression of transcripts or disease-associated mutations in living cells. We show that SHERLOCK is a versatile and robust method for detect RNA and DNA, suitable for rapid diagnostics, including infectious disease applications and sensitive genotyping. A SHERLOCK paper test can be re-engineered and synthesized in a matter of days for as little as $0.61/test (see also above “SHERLOCK is a affordable and adaptable CRISPR-Dx platform”) with confidence, as nearly all CrRNAs tested result in high sensitivity and specificity. These qualities underscore the power of CRISPR-Dx and open new avenues for the rapid, robust and sensitive detection of biological molecules. Table 13: RPA primers used Name Sequence 1st Fig. RPO683 - RPA AATTCTAATACGACTCACTATAGGGATCCTCTAGAAA SSDNA/sSRNA 1 TATGGATTACTTGGTAGAACAG (SEQ. ID No. Fig. F 18) 27B RPOC84 - RPA — |GATAAACACAGGAAACAGCTATGACCATGATTACG SSDNA/sSSRNA 1 Fig. ID R (SEQ. No. 19) 27B, AAT TCT AAT ACG ACT CAC TAT AMPL-25 Zika — |AGGCCGCTGCTAATGATAGGTTGCTACTCACAA 8B long-rpa3i- Fig. f (SEQ. ID No. 20) 31B AMPL-26 Zika — |TCAATGTCAGTCACCACTATTCCATCCACAACAG 8B long-rpa3i- Fig r (SEQ ID No. 21) 31B gaaatTAATACGACTCACTATAGGGCGTGGCGCACTA
[0497] [0497] Lateral flow based technology has achieved wide adoption in care environments due to its visual readability and detection speed. We developed a system to couple RNase activity with the release of a reporter immobilized by beads containing FAM and biotin, allowing detection in commercial lateral flow lanes (Fig. 87A). We found that this approach can reliably detect SHERLOCK activity, albeit with reduced sensitivity compared to fluorescence-based readings (Fig. 87B, C).
[0498] [0498] We designed an alternative lateral flow read based on the destruction rather than the release of a FAM-biotin reporter. An abundant reporter would accumulate the colorimetric anti-FAM antibody in the first row of the strip,
[0499] [0499] To further demonstrate the utility of SHERLOCK lateral flow, we applied the system in agricultural and health-related biotechnology scenarios. The detection of genetically modified soy is important from a regulatory point of view and for companies to monitor the use of beans. We designed a SHERLOCK assay to genotype the CP4-EPSPS gene, a herbicide-tolerant form of the 5-enolpyruvulshikimate-3-phosphate synthase from the Agrobacterium tumefaciens CP4 strain, which makes modified plants resistant to the Roundup herbicide. We designed CrRNAsS that detect CP4-EPSPS or lectin, a gene present in wild-type soybeans, and collected DNA from Roundup Ready soybeans and wild-type soybeans, using a rapid crude extraction protocol that takes less than 5 minutes. We found that SHERLOCK was able to successfully genotype RR beans with very little raw extraction history in a fast time period (-20 minutes). Furthermore, using quantitative SHERLOCK, it was possible to accurately predict the percentage of RR grains in a mixture of wild and RR grains. As GMO detection would be more applicable as a point-of-care technology in the field, we adapted the SHERLOCK assay for lateral flow and found that we could sensitively genotype grains on the paper strips using a visual readout. In addition, the lateral flow reading was readily detectable with a total incubation time of 30 minutes, allowing robust detection of SHERLOCK visually on paper (Fig. 89).
[0500] [0500] With SHERLOCKvl, we validated the technology on cell-free DNA patterns to show detection of mutations in cancer. With SHERLOCKv2, we were interested in detecting cancer mutations in patient blood samples, which is difficult because the cfDNA is usually at very low concentrations - lng/uL and the actual mutation to be detected is a small fraction of that 0.1 %-5%. We designed a SHERLOCK assay to detect the EGFR L858R mutation and cfDNA isolated from a patient carrying the mutation and a patient free of the mutation. We found that SHERLOCK was able to successfully detect the mutation (Fig. 86G) and that detection could also be performed using the lateral flow paper strips with a visual readout (Fig. 86H, 1). We also designed a SHERLOCK assay to detect a typical EGFR exon 19 deletion (5 amino acids) involved in lung cancer and found that SHERLOCK could sensibly detect this genomic change by fluorescence (Fig. 86J and Fig. 90A) and in the fluorescence strips. lateral flow (Fig. 86K, L and Fig. 90B, C).
[0501] [0501] Expression and purification of LwaCasl3a were performed as described previously ( Gootenberg et al. Science 356:438-442 (2017 )). The PsmCas13b and Csm6 orthologs were expressed and purified with a modified protocol. Briefly, bacterial expression vectors were transformed into competent Rosetta" 2 (DE3) pLysS Singles (Millipore) cells. A 12.5 mL starter culture was grown overnight in Terrific Broth 4 growth medium (Sigma) (TB ), which was used to inoculate 4 L of TB for growth at 37°C and 300 RPM to an OD600 of 0.5. At this time, protein expression was induced by supplementation with IPTG (Sigma) to a final concentration of 500 uM, and cells were cooled to 18°C for 16 h for protein expression.The cells were then centrifuged at 5000g for 15 min at 4°C. The cell pellet was harvested and stored at -80°C for further purification.
[0502] [0502] All subsequent steps of protein purification were performed at 4°C. The cell pellet was crushed and resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, pH 8.0) supplemented with protease inhibitors (complete Ultra EDTA-free tablets) lysozyme (500 ug/l ml ) and benzonase followed by high pressure cell disruption using the LM20 Microfluidizer system at 27,000 PSI. The lysate was removed by centrifugation for 1 hour at 4°C at 10,000 g. The supernatant was applied to 5 ml of StrepTactin Sepharose (GE) and incubated with rotation for 1 hour followed by washing the protein-bound StrepTactin resin three times in lysis buffer. The resin was resuspended in SUMO digestion buffer (30 mM Tris-HCl, 500 mM NaCl 1 mM DTT, 0.15% Igepal (NP-40), pH 8.0), along with 250 SUMO protease units (250 mg /ml) and incubated overnight at 4°C with rotation. The suspension was applied to a column for elution and separation of the resin by gravity flow. The resin was washed twice with 1 column volume of Lysis buffer to maximize protein elution. The eluate was diluted in cation exchange buffer (20 mM HEPES, DTT
[0503] [0503] For cation exchange and purification by gel filtration, the protein was loaded onto a 5 mL HiTrap SP HP cation exchange column (GE Healthcare Life Sciences) via FPLC (AKTA PURE, GE Healthcare Life Sciences and eluted over a gradient of 250 mM salt salt to 2M NaCl in elution buffer (20 mM HEPES, 1 mM DTT, 5% glycerol, pH 7.0) The resulting fractions were tested for the presence of recombinant protein by SDS-PAGE, and fractions containing the protein were pooled and concentrated through a Filter Centrifuge Unit (Millipore 50MWCO) to 1 ml in S200 buffer (10 mM HEPES, 1 M NaCl, 5 mM Mgcl 2 , 2 mM DIT, pH 7.0). Concentrated protein was loaded onto a gel filtration column (Superdex&o 200 Magnification 10/300 GL, GE Healthcare Life Sciences) via FPLC. The resulting gel filtration fractions were analyzed by SDS-PAGE and the protein-containing fractions were pooled and the caps changed in Storage Buffer (600 mM NaCl, 50 mM Tris-HCl pH 7.5, gly 5% cerol, 2 mM DIT) and frozen at -80°C for storage.
[0504] [0504] Accession numbers and plasmid maps for all proteins purified in this study are available in Table 17.
[0505] [0505] Rapid nucleic acid extraction was performed as described above (Wang et al. Anal Chem 89:4413-4418 (2017)). Briefly, 20 mg of ground soybeans were added to 200 µl of extraction buffer (500 mM NaoH and 10 mM EDTA), vortexed for 5 seconds and incubated for 1 minute at room temperature. After a 1:10 dilution of the supernatant, 0.4 µl of extracted genomic DNA was added to a 20 µl RPA reaction and used for SHERLOCK.
[0506] [0506] For the preparation of crRNAs, the constructs were ordered as ultrameric DNA (Integrated DNA Technologies) with a T7 promoter sequence attached. CcrRNA DNA was annealed in a short T7 primer (final concentrations 10 uM) and incubated with T7 polymerase overnight at 37°C using the HiScribe T7 Quick High Kit.
[0507] [0507] All crRNA sequences used in this study are available in Table 18. Shown is SEQ ID NO: 433441, with the complete crRNA sequence represented by SEQ ID NO: 433, the spacer sequence represented by SEQ ID NO: 434 and the direct repetition represented by SEQ ID NO: 435. The other sequence identifiers follow the same pattern.
[0508] [0508] Primers for RPA were designed using the NCBI Primer-BLAST™ using standard parameters, with the exception of amplicon size (between 100 and 140 nt), primer melting temperatures (between 54°C and 67°C) and primer size (between 30 and 35 nt). The primers were then ordered as DNA (Integrated DNA Technologies).
[0509] [0509] The RPA reactions performed were as instructed by TwistAampo Basic (TwistDx), with the exception that 280 mM MgAc was added before the input model. Reactions were performed with 1 µL input for 10 minutes at 37°C, unless otherwise noted.
[0510] [0510] For SHERLOCK quantification of nucleic acid, the concentration of the RPA primer was tested at a lower concentration of 240 nM.
[0511] [0511] When multiple targets were amplified with RPA, the primer concentration was adjusted to a final concentration of 480nM. That is, 120 nM of each primer was added to two pairs of primers for duplex detection.
[0512] [0512] All RPA primers used in this study are available in Table 19. Shown are SEQ ID NO: 442-447, with the forward primer sequences represented by SEQ ID NO: 442 and 445, the forward primer sequence with the T7RNAP promoter sequences represented by SEQ ID NOs: 443 and 446, and the reverse primer sequence represented by SEQ ID NOs: 444 and 447.
[0513] [0513] Table 19. RPA initiators used in this study.
[0514] [0514] Detection assays were performed with 45 nM purified Casl13, 22.5 nM crRNA, quenched fluorescent RNA reporter (125nM RNAse Alert v2, Thermo Scientific, homopolymer and dinucleotide (IDT) reporters); 250nM for reporter polyA Trilink), 0.5 µl murine RNase inhibitor (New England Biolabs), 25 ng background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target unless indicated otherwise, in nuclease assay buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCl2, pH 6.8). Reactions were allowed to continue for 30 min to 3 hours at 37°C (unless otherwise indicated) in a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.
[0515] [0515] All cleavage reporters used in this study are available in Table 20. SEQ ID NO: 448-451 is shown.
[0516] [0516] Detection assays were performed with 45 nM purified Casl13, 22.5 nM crRNA, quenched fluorescent RNA reporter (125nM RNAse Alert v2 and 250nM for poly A Trilink reporter), 0.5 uLl of murine RNase inhibitor (New England Biolabs), 25 ng of total human background RNA (purified from HEK293FT culture) and 1 ul of RPA reaction in nuclease assay buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCl>, pH 6 .8), rNTP mixture (11 mM final, NEB), 0.6 µl T7 polymerase (Lucigen) and 3 mM M9gCl>. Reactions were allowed to continue for 30 min to 3 hours at 37°C (unless otherwise indicated) in a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.
[0517] [0517] Casl3-Csm6 combined fluorescent cleavage assays were performed as described for standard Casl3 fluorescent cleavage reactions with the following modifications. Csm6 protein was added to the final concentration of 10 nM, 400 nM Csm6 fluorescent reporter and 500 nM Csm6 activator, unless otherwise indicated. Due to the interference of rNTPs on Csm6 activity, IVT was performed in the pre-amplification step of RPA and then 1 pL of this reaction was added as input to the Casl13-Csm6 cleavage assay.
[0518] [0518] All Csm6 activators used in this study are available in Table 21 (SEQ ID NO: 452).
[0519] [0519] For lateral flow detection, RPA was run for 10 minutes and SHERLOCK-LwaCasl3a reactions were run for 20 minutes unless otherwise noted, and the reaction was set up as noted above except with the reporter fluorescent tube replaced by final concentration luM of FAM-Biotin RNA Reporter. After incubation, the entire reaction of 20 µl of LwaCasl3a was added to 100 µl of HybriDetect 1 Assay Buffer (Milenia) and run on HybriDetect 1 side flow strips (Milenia).
[0520] [0520] SHERLOCK takes advantage of the conditional activity of the promiscuous RNase of Casl3, known as a side effect (Abudayyeh et al. Science 353, aaf5573, doi:
[0521] [0521] The detection of soybean traits is important for the worldwide surveillance of GMO traits in the food supply, and several detection methods have been developed for the detection of the most common trait, the Roundup Ready (RR) resistance gene ( Wang et al.
[0522] [0522] Simultaneous detection of lectin or other scavenger genes is important as a positive control and for loading normalization, but it is inconvenient to run a reaction for each individual crRNA, particularly in cases where the amount of sample is limiting or the content of DNA varies between aliquots. By characterizing the base cleavage preferences of the Casl3 orthologs, we found orthologs with mutually exclusive base preferences, allowing collateral cleavage to be measured by orthogonal reporters in different spectral channels (Gootenberg et al.) (Fig. 91D). Therefore, we developed an assay around LwaCasl3a using a polyuridine RNA reporter and PsmCasl3b using a polyadenine reporter. Using a LwaCasl3a crRNA complementary to the CP4 EPSPS gene and a PsmCasl3b crRNA against the lectin gene,
[0523] [0523] In many field applications, instrumentation may not be available to read a fluorescent signal. For easier visual detection, we created a reporter in SHERLOCKv2 (Gootenberg et al.) to be compatible with lateral flow lane-based reads by replacing the quenched fluorescent RNA reporter with an RNA functionalized with biotin and FAM at opposite ends (Fig. .91F). In the absence of reporter RNA cleavage, the reporter RNA is adsorbed onto a streptavidin line and captures anti-FAM antibodies labeled with gold nanoparticles. If the RNA reporter is destroyed by the side effect, the antibody will flow into a second capture line. To demonstrate this concept with rapid detection of RR soybean, we pre-amplified the CP4 EPSPS transgene with RPA from crude soybean extract in 10 minutes and then performed a LwaCasl3a detection reaction with the lateral flow RNA reporter in 20 minutes. minutes, resulting only in lateral flow signal in DNA from RR transgenic seeds (Fig. 91G, H).
[0524] [0524] We also found that signal detection of the CP4 EPSPS gene can be improved by -3x by combining the type III CRISPR-associated Csm6 endoribonuclease (Kazlauskiene et al. Science 357:605-609 (2017); Niewoehner et al. Nature 548 :543-548 92017)) in the SHERLOCK reaction (Gootenberg et al.) (Fig. 94). Using the collateral activity of LwaCasl3a to generate a hexadenylate substrate with a 2', 3rd cyclic phosphate to stimulate Csm6 cleavage activity, we can activate EiCsm6 and LsCsm6 to cause signal amplification and therefore greater signal detection in the SHERLOCK assay (Fig. 94).
[0525] [0525] In summary, SHERLOCK technology provides a useful platform for many biotechnological and agricultural applications, including surveillance of GMO traces worldwide and rapid and early detection of plant pathogens or pests.
[0526] [0526] Another goal of SHERLOCK was to engineer a visual readout of activity that does not require additional instrumentation. The Applicant first tested a colorimetric RNase reporter based on the breakdown of gold nanoparticle clusters (20, 21), but reading in this specific context required a level of RNase activity beyond what the Casl3 collateral activity was able to achieve. (FIG. 95). The Applicant then designed a lateral flow readout that was based on the destruction of a FAM-biotin reporter, allowing detection in commercial lateral flow lanes. An abundant reporter accumulates anti-FAM antibody-gold nanoparticle conjugates in the first row of the strip, preventing the binding of antibody-gold conjugates to protein A in the second row; reporter cleavage would reduce accumulation on the first line and result in signal on the second line (FIG. 96). We tested this design for detection of ZIKV or DENV ssRNA without instruments and found that detection was possible in less than 90 minutes with sensitivities up to the 2 aM condition (FIG. 9% and FIG. 97). Furthermore, the Applicant found that they could rapidly extract genomic DNA from human saliva (<10 min) and insert it directly into SHERLOCK without purification for rapid genotyping in less than 23 minutes by fluorescence and 2 hours by lateral flow (FIG 98). This exemplifies a closed-tube assay format, with the entire SHERLOCK reaction being performed in a one-pot assay, without sample purification.
[0527] [0527] The Applicant also applied the system to create a rapid, portable paper-based test for detecting mutations in liquid biopsies from non-small cell lung cancer (NSCLC) patients. The Applicant designed SHERLOCK assays to detect the EGFR L858R mutation or exon 19 deletion (5 amino acids) and cfDNA isolated from patients with or without these mutations (FIG. 96), as verified by targeted sequencing (Table 28). SHERLOCK successfully detected these mutations with both a fluorescence-based readout (FIG. 96) and a flow-based lateral readout (FIG. 96 and FIG. 99). Fluorescence-based SHERLOCK was also able to detect a common mutation other than EGFR, T790M, in synthetic liquid biopsy and CfDNA patient samples (FIG. 99 (e) (£)).
[0528] [0528] To improve detection robustness and reduce the probability of false positive reading, we combined Csm6 with Casl3 detection in lateral flow (FIG. 96). We tested lateral flow reporters of various sequences and lengths in the presence of Csm6 and activator and found that a long A-C reporter demonstrated a strong cleavage signal (FIG. 100A, B). We used this reporter in combination with the lateral flow reporter Casl13 for rapid detection of DENV ssRNA, relying only on Csm6 for amplification (ie, in the absence of RPA) ( FIG. 96 (L)). Subsequently, we combined the reading of RPA, Casl3/Csm6 and lateral flow to detect an acyltransferase target and found that the increase in signal imparted by Csm6 allowed for faster detection by lateral flow (FIG. 100C-D) with reduced background.
[0529] [0529] Expression and purification of LwaCasl3a were performed as described previously (3) with minor modifications and is detailed below. The orthologs LbuCasl3a, LbaCasl3a, Casl3b and Csm6 were expressed and purified with a modified protocol. Briefly, bacterial expression vectors were transformed into competent RosettaTM 2(DE3)pLysS Singles (Millipore) cells. A 12.5 mL starter culture was grown overnight in Terrific Broth 4 (Sigma) growth medium (TB), which was used to inoculate 4 L of TB for growth at 37°C and 300 RPM to an OD600 of 0.5 . At this time, protein expression was induced by supplementation with IPTG (Sigma) to a final concentration of 500 uM, and the cells were cooled to 18ºC for 16 h for protein expression. The cells were then centrifuged at 5000g for 15 min at 4°C. The cell pellet was harvested and stored at -80°C for further purification.
[0530] [0530] All subsequent steps of protein purification were performed at 4°C. The cell pellet was crushed and resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, pH 8.0) supplemented with protease inhibitors (complete tablets without Ultra EDTA),
[0531] [0531] For cation exchange and gel filtration purification, the protein was loaded onto a 5 mL HiTrap SP HP cation exchange column (GE Healthcare Life Sciences) via FPLC (AKTA PURE, )
[0532] [0532] GE Healthcare Life Sciences) and eluted over a salt gradient from 250 mM to 2M NaCl in elution buffer (20 mM HEPES, 1 mM DTT, 5% glycerol, pH 7.0; pH 7.5 for LbuCasl3a , LbaCasl3a). The resulting fractions were tested for the presence of recombinant protein by SDS-PAGE, and the fractions containing the protein were pooled and concentrated through a Centrifugal Filter Unit (Millipore 50MWCO) to 1 mL in S200 buffer (mM HEPES, 1 M NaCl , 5 mM MgCl 2 , 2 mM DTIT, pH 7.0). The concentrated protein was loaded onto a gel filtration column (Superdexo 200 Magnification 10/300 GL, GE Healthcare Life Sciences) via FPLC. Fractions resulting from gel filtration were analyzed by SDS-PAGE and fractions containing protein were pooled and caps exchanged in Storage Buffer (600 mM NaCl, 50 mM Tris-HCl pH 7.5, 5% glycerol, DIT 2 mM) and frozen at -80°C for storage.
[0533] [0533] Accession numbers and plasmid maps for all proteins purified in this study are available in Table 22.
[0534] [0534] Nucleic acid targets for Casl2a and genomic DNA detection were PCR amplified with NEBNext PCR master mix, gel extracted and purified using the MinElute Gel Extraction Kit (Qiagen). For RNA-based detection, purified dsDNA was incubated with T7 polymerase overnight at 30°C using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) and the RNA was purified with the MEGAClear Transcription Clean-up Kit (Thermo Fisher
[0535] [0535] The crRNA preparation was performed as described previously (3) with minor modifications and is detailed below. For the preparation of crRNAs, the constructs were ordered as ultrameric DNA (Integrated DNA Technologies) with a T7 promoter sequence attached. CcrRNA DNA was annealed in a short T7 primer (final concentrations 10 uM) and incubated with T7 polymerase overnight at 37°C using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs). crRNAs were purified using clean RNAXP beads (Beckman Coulter) at 2x bead to reaction volume ratio, with an additional 1.8x supplementation of isopropanol (Sigma).
[0536] [0536] All crRNA sequences used in this study are available in Table 23. Table 23 lists SEQ ID NO: 453-827, with SEQ ID NO: 453 representing the complete crRNA sequence, SEQ ID NO: 454 representing the spacer and SEQ ID NO: 455 representing the direct repeat for LwaCasl3a. The other sequence identifiers in the table follow the same pattern. All DNA and RNA target sequences used in this study are available in Table 24.
[0537] [0537] The primers for RPA were designed using the NCBI Primer-BLAST(27) using standard parameters, with the exception of amplicon size (between 100 and 140 nt), primer melting temperatures (between 54°C and 67°C), and initiator (between 30 and 35 nt). The primers were then ordered as DNA (Integrated DNA Technologies).
[0538] [0538] The RPA and RT-RPA reactions performed were as instructed by TwistAmpo Basic or TwistAmpEo Basic RT (TwistDx), respectively, with the exception that 280 mM MgAc was added before the input model. Reactions were performed with 1 npL of input for 1 hour at 37°C, unless otherwise noted.
[0539] [0539] For SHERLOCK nucleic acid quantification, RPA primer concentration was tested at standard (480nM final) and lower (240nM, 120nM, 60nM, 24nM) concentration to find the optimal concentration. RPA reactions were carried out for 20 minutes.
[0540] [0540] When multiple targets were amplified with RPA, the primer concentration was adjusted to a final concentration of 480nM. That is, 120 nM of each primer was added to two pairs of primers for duplex detection.
[0541] [0541] All RPA primers used in this study are available in Table 25. Shown are SEQ ID NO: 841-870, with SEQ ID NO: 841 representing the forward primer sequence, SEQ ID NO: 842 representing the forward primer sequence with the T7 RNAP promoter and SEQ ID NO: 843 representing the reverse primer sequence for DENV ssRNA. The other sequence identifiers follow the same pattern.
[0542] [0542] The detection assays were performed as described above (3) with minor modifications and the procedure is detailed below. Detection assays were performed with 45 nM purified Casl3, 22.5 nM crRNA, quenched fluorescent RNA reporter (125nM RNAse Alert v2, Thermo
[0543] [0543] Scientific, Homopolymer and Dinucleotide (IDT) reporters; 250nM for Trilink polyA reporter), 0.5 µl murine RNase inhibitor (New England Biolabs), ng background total human RNA (purified from HEK293FT culture), and varying amounts of incoming nucleic acid target unless otherwise indicated, in nuclease assay buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCl 2 , pH 6.8). For Csm6 fluorescent cleavage reactions, the protein was used at a final concentration of 10nM, along with 500nM of 2',3' cyclic phosphate oligoadenylate, 250nM of fluorescent reporter, and 0.5 µl of murine RNase inhibitor in assay buffer. nuclease (20 mM HEPES, 60 mM NaCl, 6 mM MgCl 2 , pH 6.8). Reactions were allowed to continue for 1-3 h at 37°C (unless otherwise indicated) in a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min. In reactions involving AsCasl2a, 45nM AsCasl2a was included using the IDT recombinant protein. In the case of multiplexed reactions, 45nM of each protein and 22.5nM of each crRNA were used in the reaction.
[0544] [0544] All cleavage reporters used in this study are available in Table 26. Shown are SEQ ID NOs: 871-877, representing sequences of 10 nucleotides in length or more. Sequences “less than 10 nucleotides were not designated as sequence identifiers.
[0545] [0545] Detection assays were performed with 45 nM purified Casl13, 22.5 nM crRNA, quenched fluorescent RNA reporter (125nM RNAse Alert v2, Thermo Scientific, homopolymer and dinucleotide (IDT) reporters), 250nM for reporter polyA Trilink), 0.5 npL of murine RNase inhibitor (New England Biolabs), 25 ng of background total human RNA (purified from HEK293FT culture), and 1 ul of RPA reaction in nuclease assay buffer (20 mM HEPES, 60 mM NaCl, 6 mM MgCl2, pH 6.8), rNTP mixture (final ImMM, NEB), 0.6 ul T7 polymerase (Lucigen) and 3mMM MgCl2. Reactions were allowed to continue for 1-3 h at 37°C (unless otherwise indicated) in a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.
[0546] [0546] For the detection of one-pot nucleic acid, the detection assay was performed as described above (3) with minor modifications. A single 100 ul pool reaction assay consisted of 0.48 uM forward primer, 0.48 uM reverse primer, 1x RPA rehydration buffer, varying amounts of DNA input, 45 nM LwCasl3a recombinant protein, 22 .5 nM crRNA, 125 ng human RNA total background, 125 nM substrate reporter (RNase alert v2), 2.5 µl murine RNase inhibitor (New England Biolabs), 2 mM ATP, 2 mM GTP, 2 mM UTP, 2 mM CTP, 1 µl T7 polymerase mix (Lucigen), 5 mM M9gCl 2 , and 14 mM MgAc. Reactions were allowed to continue for 1-3 h at 37°C (unless otherwise indicated) in a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min. For the lateral flow reading, 20 µl was added. reaction to 100 µl of HybriDetect 1 Assay Buffer (Milenia) and run on HybriDetect 1 (Milenia) side flow strips.
[0547] [0547] Target RNA was transcribed in vitro from a dsDNA template and purified as described above. The in vitro cleavage reaction was performed as described above for the fluorescence cleavage reaction with the following modifications. The fluorescence reporter was replaced by lug target RNA and no background RNA was used. The cleavage reaction was carried out for 5 minutes (LwaCasl3a) or 1 hour (PsmCasl3b) at 37ºC. The cleavage reaction was purified using the RNA clean & concentrator-5 kit (Zymo Research) and eluted in 10 µl of UltraPure water (Gibco). The cleavage reaction was further labeled with 10pug of maleimide IRDye 800CW (Licor), following the protocol of the 5'EndTag labeling reaction kit (Vector Laboratories). To determine the 5' end produced by the Casl3 cleavage, the protocol was modified to perform an alkaline phosphatase (AP) treatment or replace the UltraPure water to label only 5'-OH-containing RNA species, while triphosphorylated (PPP) RNA species undigested are scored only when AP treatment is performed.
[0548] [0548] To determine the cleavage ends produced by the collateral RNase activity of Casl3 by Mass Spectrometry, an in vitro cleavage reaction was performed as described above with the following modifications. The Casl3 RNA target was used at a final concentration of 1 nM, the activator Csm6 at a final concentration of 3pM and no background RNA was used. For control reactions, the Casl13 target was replaced with UltraPure water or the standard in vitro cleavage reaction was incubated with hexa-adenylate containing a 2' and 3' cyclic phosphate activator in the absence of Casl3 target, Casl13 protein and Casl3 crRNA . Cleavage reactions were performed for 1 hour at 37°C and purified using a New England Biolabs siRNA purification protocol. Briefly, a tenth volume of 3 M NaOAc, 2 ul of RNase-free Glicoblue (Termofisher) and three volumes of cold 95% ethanol were added, placed at -20°C for 2 hours and centrifuged for 15 minutes at 14,000 g. The supernatant was removed and two volumes of 80% EtOH were added and incubated for 10 minutes at room temperature. The supernatant was decanted and the samples centrifuged for 5 minutes at
[0549] [0549] For mass spectrometry analysis, samples were diluted 1:1 with UltraGrade water and analyzed on the Bruker Impact II q-TOF mass spectrometer in negative ion mode coupled to an Agilent 1290 HPLC. PLRP-S (50 mm, 5 µm particle size, 1000 angstrom pore size PLRP-S column, 2.1 mm ID) using 0.1% v/v ammonium hydroxide in water as mobile phase A and acetonitrile as mobile phase B. The flow rate was kept constant over 0.3 ml/minute. Mobile phase composition started at 0% B and was maintained for the first 2 minutes. After that point, the composition was changed to 100% B for the next 8 minutes and held for one minute. The composition was then returned to 0% B over 0.1 minute and then held for the next 4.9 minutes to allow the column to re-equilibrate to initial conditions. The mass spectrometer was adjusted for large MW ions and data were acquired between m/z 400-5000. The entire mass spectrometer dataset was m/z calibrated using a sodium formate injection. Data were analyzed using Bruker Compass Data Analysis 4.3 with a license to the MaxEnt deconvolution algorithm to generate a neutral mass spectrum calculated from the negatively charged ion data.
[0550] [0550] DNA extraction from saliva was performed as described above (3) with minor modifications and is detailed below. 2mlLl of saliva were collected from volunteers who were prevented from consuming food or drinks 30 min before collection. The samples were then processed using the QIAampê DNA Blood Mini Kit (Qiagen) as recommended by the kit protocol. For boiled saliva samples, 400 µl of phosphate-buffered saline (Sigma) was added to 100 µl of volunteer saliva and centrifuged for 5 min at 1800 g. The supernatant was decanted and the pellet was resuspended in phosphate-buffered saline with 0.2% Triton X-100 (Sigma) before incubation at 95°C for 5 min. 1 uL of the sample was used as a direct input in the RPA reactions.
[0551] [0551] ddPCR quantification was performed as described previously (3) with minor modifications and is detailed below. To confirm the concentration of the target dilutions, we performed PCR with digital droplets (dAddPCR). For DNA quantification, droplets were made using the ddPCR Supermix for Probes (dUTP-free (BioRad) with PrimeTime qPCR probes/primer assays (IDT) designed for the target sequence. For RNA quantification, droplets were made using the one-step RT-ddPCR kit for probes with PrimeTime qgPCR probes/primer assays designed for the target sequence. Droplets were generated in both cases using the QX200 droplet generator (BioRad) and transferred to a PCR plate. in droplets was performed in a thermocycler as described in the kit protocol, and nucleic acid concentrations were subsequently determined by measurement on a QX200 droplet reader.
[0552] [0552] Casl3-Csm6 combined fluorescent cleavage assays were performed as described for standard Casl3 fluorescent cleavage reactions with the following modifications. Csm6 protein was added to the final concentration of 10 nM, 400 nM Csm6 fluorescent reporter and 500 nM Csm6 activator, unless otherwise indicated. To distinguish Casl3 from the collateral RNase activity of Csm6, two distinct fluorophores were used for fluorescence detection (FAM and HEX). Due to the interference of rNTPs on Csm6 activity, IVT was performed in the pre-amplification step of RPA and then 1 pL of this reaction was added as input to the Casl13-Csm6 cleavage assay.
[0553] [0553] In the case where we tested a three-step Casl3-Csm6 cleavage assay, the RPA was performed normally as discussed above for varying periods and then used as input to a normal IVT reaction for varying periods. Next, IVT's lulLl was used as input for the Casl3-Csm6 reaction described in the previous paragraph. All Csm6 activators used in this study are available in Table 27.
[0554] [0554] To track Cas13 cleavage preference, an in vitro RNA cleavage reaction was established as described above with the following modifications. The Casl3 target was used at 20nM, the fluorescent reporter was replaced by 1 uM DNA-RNA oligonucleotide (IDT) containing a 6-mer stretch of randomized ribonucleotides flanked by DNA loops for the preparation of the NGS library. Reactions were carried out for 60 minutes (unless otherwise indicated) at 37°C. Reactions were purified using the Zymo oligo-clean kit and concentrator-5 (Zymo research) and 15pL of UltraPure water was used for elution. 10uL of purified reaction was used for reverse transcription using a gene-specific primer that binds to the DNA identifier.
[0555] [0555] Reverse transcription (RT) was performed for 45 minutes at 42ºC, according to the qaScript Flex CDNA-kit protocol (quantabio). To assess cleavage efficiency and product purity, RT reactions were diluted 1:10 in water and loaded into a Small RNA kit and run on a Bioanalyzer 2100 (Agilent). Four microliters of RT reaction were used for the first round of NGS library preparation. NEBNext (NEB) was used to amplify first strand CDNA with a mixture of forward primers at the end of 625 nM and a reverse primer at 625 nM for 15 cycles with initial denaturation of 3 minutes at 98°C, cycle denaturation from 10s to 98ºC, 10s annealing at 63ºC, 20s extension at 72ºC and 2 minutes final extension at 72ºC.
[0556] [0556] Two microliters of the first round PCR reaction were used for second round PCR amplification to attach Illumina compatible indices (NEB) for NGS sequencing. The same NEBNext PCR protocol was used for amplification. PCR products were analyzed by agarose gel electrophoresis (2% Sybr Gold E-Gel Invitrogen system) and 5pL of each reaction was pooled. The pooled samples were gel extracted, quantified with a high-sensitivity Qubit DNA 2.0 DNA kit and normalized to a final concentration of 4 nM. The final library was diluted to 2 pM and sequenced on a NextSeq 500 Illumina system using a 75 cycle kit.
[0557] [0557] To analyze the depletion of preferred motifs from the random motif library screen, 6-mer regions were extracted from the sequence data and normalized to the overall read count for each sample. The normalized read counts were then used to generate log ratios, with pseudocount adjustment, between experimental conditions and corresponding controls. For Casl3 experiments, the corresponding controls did not have target RNA added; for experiments with Csm6 and RNase A, matched controls lacked enzyme. The log ratio distribution format was used to determine cutoff points for enriched motives. Enriched motifs were then used to determine the occurrence of 1, 2 or 3 nucleotide combinations. Motif logos were generated using Weblogo3 (26).
[0558] [0558] To study ortholog clustering, several sequence alignments were generated with the Casl3a and Casl3b protein sequences in Geneious with MUSCLE and then clustered using the Euclidean distance in R with the heat map.2 Function. To study direct repeat clustering, alignments of several sequences were generated with the direct repeating sequences Casl3a and Casl3b in Geneious using the Geneious algorithm and then clustered using the Euclidean distance in R with the heat map.2 Function. To study clustering of orthologs based on nucleotide motif preference, the cleavage activity matrix was clustered using the Euclidean distance in R using the heat map.2 Function.
[0559] [0559] An RNA oligo was synthesized from the IDT with thiols at the 5' and 3' ends (Table 26 for sequence). To deprotect the thiol groups, the oligo to a final concentration of 20 mM was reduced in 150 mM sodium phosphate buffer containing 100 mM DTT for 2 hours at room temperature. The oligos were then purified using NAP-5 sephadex columns (GE Healthcare) in a final volume of 700uL of water. As described previously (20), the 10UuM reduced oligo was added to a volume of 280puL to 600pL of 2.32nM gold and 15nm nanoparticles (Ted Pella), which is a 2000:1 ratio of oligo to nanoparticles. Subsequently, 10uL of 1M Tris-HCl at pH8.3 and 90pL of 1M NaCl were added to the mixture of oligo-r nanoparticles and incubated for 18 hours at room temperature with rotation. After 18 hours, additional 1M Tris-HCl (5UL at pH8.3) was added with 5M NaCl (50uL) and this was incubated for an additional 15 hours at room temperature with rotation. After incubation, the final solution was centrifuged for 25 min at 22,000 g. The supernatant was discarded and the conjugated nanoparticles were resuspended in 50uL of 200mM NaCl.
[0560] [0560] The nanoparticles were tested for RNase sensitivity using an RNase A assay. Varying amounts of RNase A (Thermo Fischer) were added to 1x RNase A buffer and 6uL of conjugated nanoparticles in a total reaction volume of 20uL. Absorbance at 520 nm was monitored every 5 minutes for 3 hours using a plate spectrophotometer.
[0561] [0561] For lateral flow based on cleavage of a FAM-RNA-biotin reporter, non-RPA LwaCasl3a or SHERLOCK-LwaCasl3a reactions were run for 1 hour, unless otherwise noted, with final luM concentration of FAM reporter -RNA-biotin. After incubation, 20 µl of LwaCasl3a supernatant reactions were added to 100 µl of HybriDetect 1 assay buffer (Milenia) and run on HybriDetect 1 side flow strips (Milenia).
[0562] [0562] Cloning of REPAIR constructs, transfection of mammalian cells, isolation of RNA and preparation of the NGS library for REPAIR
[0563] [0563] Constructs to simulate the reversion of APC mutations and guide constructs for REPAIR were cloned as described previously (23). Briefly, 96 nt sequences centered on the APC:cC.1262G>A mutation were designed and the golden gate was cloned under an expression vector, and the corresponding guide sequences were the golden gates cloned into the U6 expression vectors for the PspCas13b guides. To simulate patient samples, 300 ng of wild-type or mutant APC expression vector was transfected into HEK293FT cells with Lipofectamine 2000 (Invitrogen), and two days after transfection, post-transfection DNA was collected with the Qiamp DNA Blood Kit. Midi (Qiagen), following the manufacturer's instructions. 20 ng of DNA was used as input in the SHERLOCK-LwaCasl3a reactions.
[0564] [0564] RNA correction using the REPAIR system was performed as previously described (23): 150 ng of dPspCas13b-ADAR (DD) E488Q, 200 ng of guide vector and 30 ng of APC expression vector were co-transfected and RNA Two days after transfection, cotransfected samples were harvested with the RNeasy Plus Mini Kit (Qiagen), following the manufacturer's instructions. 30 ng of RNA was used as input in the SHERLOCK-LwaCasl3a reactions. All plasmids used for editing RNA REPAIR in this study are available in Table 29.
[0565] [0565] RNA editing fractions were independently determined by NGS as described above. RNA was reverse transcribed with the qScript Flex kit (Quanta Biosciences) with a sequence-specific primer. First strand cDNA was amplified with NEBNext High Fidelity 2X PCR Mastermix (New England Biosciences) with a mixture of forward primers at 625nM final and a reverse primer at 625nM for 15 cycles with initial denaturation of 3 minutes at 98°C, cycle denaturation of 10 seconds at 98ºC, 30 seconds annealing at 65ºC, 30 seconds extension at 72ºC and final extension extension of 2 minutes at 72ºC. Two microliters of the first round PCR reaction were used for second round PCR amplification to fix Illumina-compatible indices for NGS sequencing, with NEBNext, using the same protocol with 18 cycles. PCR products were analyzed by agarose gel electrophoresis (2% Sybr Gold E-Gel Invitrogen) and 5u1pL of each reaction was pooled. Pooled samples were gel extracted, quantified with the high-sensitivity Qubit DNA 2.0 DNA kit and normalized to a final concentration of 4nM and read with a 300 cycle MiSeq v2 kit (Illumina).
[0566] [0566] SHERLOCK fluorescence analysis was performed as described previously (3) with minor modifications and is detailed below. To calculate the background subtracted fluorescence data, the initial fluorescence of the samples was subtracted to allow comparisons between different conditions. Fluorescence for background conditions (no input or no crRNA conditions) was subtracted from the samples to generate subtracted background fluorescence.
[0567] [0567] The crRNA to SNP discrimination ratios were calculated to adjust for the overall sample-to-sample variation as follows: RNA A; r Shallow = (m + n)A; CrRNA A; Tr Ratio = SEA TELOE, TELE, where Aj and Bi refer to the SHERLOCK intensity values for the replication technique i of the crRNAs that detect the A allele or B allele, respectively, for a given individual. As we normally have four technical repeats per crRNA, m and n are equal to 4 and The denominator is equivalent to the sum of all eight SHERLOCK crRNA intensity values for a given locus and SNP and individual. Because there are two crRNAs, the average ratio of crRNAs in each of an individual's crRNAs will always add up to two. Therefore, in the ideal case of homozygosity, the average ratio of crRNA to positive CcrRNA allele will be two and the average ratio of crRNA to negative crRNA allele will be zero. In the ideal case of heterozygosity, the average ratio of CrRNA to each of the two crRNAs will be one. As in SHERLOCKv2, we perform genotyping by measuring A; and Bj in different color channels, we scale the 530 color channel by 6 to match the intensity values in the 480 color channel.
[0568] [0568] Some members of the Casl3 family, such as PinCasl3b and LbuCasl3ôa, demonstrate promiscuous cleavage in the presence or absence of a target, and this background activity is dinucleotide reporter dependent (FIG. 101). This background activity also depended on the spacer for Lbucasl3a. In some reporters, U and A base preferences clustered within protein similarity or DR. Interestingly, the dinucleotide preferences identified here did not match the clustered Casl3 families of direct repeat or protein similarity (FIG. 101).
[0569] [0569] To identify the ideal crRNA for detection with PsmCasl3b and CcaCasl3b, we tested "34 to 12 nt crRNA spacer lengths and found that PsmCasl13b had a peak sensitivity at the spacer of 30, while CcecaCas13b had equivalent sensitivity above the lengths of 28nt, justifying the use of 30nt spacers to assess the activity of Casl13 To further explore the robustness of the targeting of CcaCasl3b and PsmCasl3b compared to LwaCasl3a, we designed eleven different crRNAs evenly spaced by ssRNA 1 and found that the collateral activity of LwaCasl3a was robust for crRNA design, while CcaCasl13b and PsmCasl3b both showed more variability in activity between different crRNAs.
[0570] [0570] Random screening of library motifs for additional orthogonal motifs To further explore the diversity of cleavage preferences of the Casl3a and Casl3b orthologs, we developed a library-based approach to characterizing preferred motifs for collateral endonuclease activity. We used a 6-mer RNA degenerate reporter, flanked by constant DNA loops, which allowed amplification and reading of uncleaved sequences. Incubating this library with Casl3 enzymes resulted in detectable cleavage patterns that depended on the addition of target RNA (Fig. S12B), and sequencing of exhausted motifs from these reactions revealed an increase in library slope over digestion time, indicative of a population of preferred reasons for cleavage. Sequence logos and highly depleted base-paired preferences reproduce the U-preference observed for LwaCasl3a and CcaCasl13b and the A-preference of PsmCasl3b. We synthesized reporters of the top motives as determined on screen to validate the findings, and found that LwaCasl3a, CcaCasl3a, and PsmCasl3b cleaved their most preferred motive. We also found several sequences that showed cleavage for only one ortholog, but not for others, which could allow an alternative oorthogonal reading of dinucleotide motifs. LwaCasl3a incubated with different targets produced similar cleavage motif preferences, indicating that the basic collateral activity preference is constant, regardless of target sequence.
[0571] [0571] Using mass spectrometry, we verified that digestion with LwaCaslô3a produced the expected cyclic phosphate-terminated products for Csm6 activation. Activation was more effective for designs with 3' protection with poly U, as other activation designs, including 5' protection with internal poly WU and poly U tracts, were less effective in activating Csm6 exclusively in the presence of target RNA, probably because LwaCasl3a has little activity on UA motifs and the 5' guard is ineffective in preventing Csm6 activation.
[0572] [0572] As the combination of Csm6 enhancement with RPA pre-amplification would increase signal and sensitivity, we tested Csmb for activity in the presence of in vitro transcriptional components required for combination with RPA.
[0573] [0573] This concept involves two probes: FAM-T*A*rArUG*C*-Biotin (LwaCasl3a cuts) and FAM-T*A*rUrAG*C*t-DIG (CcaCasl13b10 cuts). These probes connect the anti-
[0574] [0574] In this assay, two probes were used: º FAM-T*A*rArUG*C*-Biotin (cuts —“LwaCasl3a) - detection of SssRNA 1 o. FAM-T*A*rUrAG*C*-DIG (CcaCasl3b10 cuts) - Dengue RNA detection Results are shown in Figures 103A and 103B.
[0575] [0575] Applicants designed and synthesized lateral flow bands that allow for 4 lines and simultaneous detection of 4 targets.
[0576] [0576] The probes used were as follows: . /S5TYE665/T*A*rArUG*C*/3AlexF488N/ - LwaCasl3a . /S5TYE665/T*A*rUrAG*C*/36-FAM/ - CcaCas13b .º /STYE665/rArArArArA/3Bio/ - PsmCasl3b .º /STYEG665/AAAAA/3Dig N/ - AsCasl2a
[0577] [0577] Strips contain anti-Alexa-fluor-488, anti-FAM, streptavidin and anti-Dig lines, allowing detection of up to 4 targets. Tye665 dye will be detected and decreases in fluorescent line intensity will indicate the presence of the target.
[0578] [0578] Additional modalities are disclosed in the following numbered paragraphs:
[0579] [0579] Various modifications and variations of the described methods, pharmaceutical compositions and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modification and that the invention, as claimed, should not be unduly limited to those specific purposes. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those in the art should be within the scope of the invention. This application is intended to cover any variations, uses or adaptations of the invention, generally following the principles of the invention and including such deviations from the present disclosure, are within the customary practice known in the art to which the invention pertains and may be applied. essential resources mentioned above.

权利要求:
Claims (75)
[1]
1. A lateral flow device characterized in that it comprises a substrate comprising a first end, wherein the first end comprises a sample loading portion and a first region loaded with a detectable ligand, a CRISPR effector system, a detection, a first capture region comprising a first binding agent and a second capture region comprising a second binding agent, wherein the CRISPR effector system comprises a CRISPR effector protein and one or more leader sequences, each leader sequence configured to bind one or more target molecules.
[2]
2. Lateral flow device, according to claim 1, characterized in that the detection construct comprises an RNA or DNA oligonucleotide, comprising a first molecule at a first end and a second molecule at a second end.
[3]
3. A lateral flow device, according to claim 1, characterized in that the sample loading portion further comprises one or more amplification reagents to amplify one or more target molecules.
[4]
4. Lateral flow device, according to claim 3, characterized in that the reagents to amplify the one or more target RNA molecules comprise nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA) , loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), PCR, multiple displacement amplification (MDA), circle amplification (RCA), ligase chain reaction (LCR) or branch amplification method (RAM).
[5]
5. Lateral flow device, according to claim 2, characterized in that the first molecule is FITC and the second molecule is biotin, or vice versa.
[6]
6. A lateral flow device according to claim 5, characterized in that the first capture region is close to and at the same end of the lateral flow substrate as the sample loading portion.
[7]
7. Lateral flow device, according to claim 5, characterized in that the first capture region comprises a first binding agent that specifically binds the first molecule of the reporter construct.
[8]
A lateral flow device according to claim 7, characterized in that the first binding agent is an antibody that is fixed or otherwise immobilized in the first capture region.
[9]
9. A lateral flow device according to claim 1, characterized in that the second capture region is located towards the opposite end of the lateral flow substrate from the first binding region.
[10]
10. Lateral flow device, according to claim 9, characterized in that the second capture region comprises a second binding agent that specifically binds the second molecule of the reporter construct, or the detectable ligand.
[11]
11. The lateral flow device of claim 10, wherein the second binding agent is an antibody or antibody-binding protein that is fixed or otherwise immobilized in the second capture region.
[12]
12. Lateral flow device, according to claim l1, characterized in that the detectable ligand is a gold nanoparticle.
[13]
13. Lateral flow device, according to claim 12, characterized in that the gold nanoparticle is modified with a binding agent that specifically binds the second molecule of the detection construct.
[14]
14. Lateral flow device, according to claim 13, characterized in that the first antibody is an anti-FITC antibody.
[15]
15. Lateral flow device, according to claim 8, characterized in that the antibody is an anti-FITC antibody.
[16]
16. Lateral flow device, according to claim 8, characterized in that the antibody is an anti-biotin antibody.
[17]
17. Lateral flow device, according to claim 1, characterized in that the substrate is a substrate of flexible materials.
[18]
18. Lateral flow device, according to claim 1, characterized in that the substrate of flexible materials is a paper substrate or a flexible polymer-based substrate.
[19]
19. Lateral flow device, according to claim 18, characterized in that the CRISPR effector protein is an RNA targeting effector protein, a DNA targeting protein or a combination thereof.
[20]
20. Lateral flow device, according to claim 19, characterized in that the RNA targeting effector protein is a Cas1l3.
[21]
21. Lateral flow device, according to claim 20, characterized in that the effector protein Casl13 is from an organism of a genus selected from the group consisting of: Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor , Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira.
[22]
22. Lateral flow device, according to claim 21, characterized in that the Casl13 effector protein is from an organism selected from the group consisting of: Leptotrichia shahii; Leptotrichia wadei (Lw2); Listeria seeligeri; Lachnospiraceae bacterium MAZ2020; Lachnospiraceae bacterium NK4A179; [Clostridium] aminophilum DSM 10710; Carnobacterium gallinarum DSM 4847; Carnobacterium gallinarum DSM 4847 (second CRISPR loci); Paludibacter propionicigenes WB4; Listeria weihenstephanensis FSL R9-0317; Listeriaceae bacterium FSL M6-0635; Leptotrichia wadei FO279; Rhodobacter capsulatus SB 1003; Rhodobacter capsulatus R121; Rhodobacter capsulatus
DE442; Leptotrichia buccalis C-1013-b; Herbinix hemicellulosilytica; [Eubacterium] rectal; Eubacteriaceae bacterium CHKCIOO04; Blautia sp. Marseille-P2398; and Leptotrichia sp. oral taxon 879 str. FO557. Twelve (12) other non-limiting examples are: Lachnospiraceae bacterium NK4A144; Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp. TSL5-11; Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautia sp. Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroides ihuae; Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; and Insolitispirillum peregrinum.
[23]
23. Lateral flow device, according to claim 22, characterized in that the effector protein Cal3 is an effector protein L. wadei F0279 or L. wadei FO0279 (Lw2) C2c2.
[24]
24. Lateral flow device, according to claim 19, characterized in that the DNA targeting effector protein is a Casl2.
[25]
25. Lateral flow device, according to claim 24, characterized in that the Cal2 is Cpfl, C2cl or a combination thereof.
[26]
26. Lateral flow device according to any one of claims 1 to 25, characterized in that the one or more guide sequences that are diagnostic for a disease state.
[27]
27. Lateral flow device, according to claim 26, characterized in that the disease state is cancer.
[28]
28. Lateral flow device, according to claim 26, characterized in that the disease state is an autoimmune disease.
[29]
29. Lateral flow device, according to claim 26, characterized in that the disease state is an infection.
[30]
30. Lateral flow device, according to claim 29, characterized in that the infection is caused by a virus, a bacterium, a fungus, a protozoan or a parasite.
[31]
31. Lateral flow device, according to claim 30, characterized in that the infection is a viral infection.
[32]
32. Lateral flow device, according to claim 31, characterized in that the viral infection is caused by a DNA virus.
[33]
33. Lateral flow device, according to claim 32, characterized in that the DNA virus is a Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus and Varicella Zoster virus), Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae,
Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fever virus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses , Polyomaviridae (including Simian virus 40, JC virus, BK virus), Poxviridae (including cowpox and smallpox), Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus.
[34]
34. Lateral flow device, according to claim 31, characterized in that the viral infection is caused by a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, a retroviruses or a combination thereof.
[35]
35. Lateral flow device according to claim 34, characterized in that the viral infection is caused by a Coronaviridae virus, Picornaviridae virus, Caliciviridae virus, Flaviviridae virus, Togaviridae virus, a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunenaviridae, a Bunyaviridae, an Orthomyxoviridae or a Deltavirus.
[36]
36. Lateral flow device, according to claim 35, characterized in that the viral infection is caused by Coronavirus, SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, yellow fever virus, West Nile virus, Hepatitis C Virus, Dengue Virus, Zika Virus, Rubella Virus, Ross River Virus, Sindbis Virus, Chikungunya Virus, Borna Disease Virus, Ebola Virus, Marburg Virus, Measles Virus, Mumps Virus, Nipah Virus, Hendra Virus , Newcastle disease virus, human respiratory syncytial virus, rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, influenza or Hepatitis D virus.
[37]
37. Lateral flow device, according to claim 30, characterized in that the infection is a bacterial infection.
[38]
38. Lateral flow device, according to claim 37, characterized in that the bacterium causing the bacterial infection is a species of Acinetobacter, a species of Actinobacillus, a species of Actinomycetes, a species of Actinomyces, a species of Aerococcus, a species of Aeromonas, a species of Anaplasma, a species of Alcaligenes, a species of Bacillus, a species of Bacteriodes, a species of Bartonella, a species of Bifidobacterium, a species of Bordetella, a species of Borrelia, a species of Brucella, a species of Burkholderia, a species of Campylobacter, a species of Capnocytophaga, a species of Chlamydia, a species of Citrobacter, a species of Coxiella, a species of Corynbacterium, a species of Clostridium, a species of Eikenella, a species of Enterobacter , a species of Escherichia, a species of Enterococcus, a species of Ehlichia, a species of Epidermophyton, a species of Erysipelothrix, a species of Eubacterium, a species of Francisella, a species of Fusobacterium, a species of Gardnerella, a species of Gemella, a species of Haemophilus, a species of Helicobacter, a species of Kingella, a species of Klebsiella, a species of Lactobacillus, a species of Lactococcus, a species of Listeria, a species of Leptospira, a species of Legionella, a species of Leptospira, a species of Leuconostoc, a species of Mannheimia, a species of Microsporum, a species of Micrococcus, a species of Moraxella, a species of Morganell, a species of Mobiluncus, a species of Micrococcus, a species of Mycobacterium, a species of Mycoplasm, a species of Nocardia, a species of Neisseria, a species of Pasteurelaa, a species of Pediococcus, a species of Peptostreptococcus, a species of Pityrosporum, a species of Plesiomonas, a species of Prevotella, a species of Porphyromonas, a species of Proteus, a species of Providencia, a species of Pseudom onas, a species of Propionibacteriums, a species of Rhodococcus, a species of Rickettsia, a species of Rhodococcus, a species of Serratia, a species of Stenotrophomonas, a species of Salmonella, a species of Serratia, a species of Shigella, a species of Staphylococcus, a species of Streptococcus, a species of Spirillum, a species of Streptobacillus, a species of Treponema, a species of Tropheryma, a species of Trichophyton, a species of Ureaplasma, a species of Veillonella, a species of Vibrio, a species of Yersinia , a species of Xanthomonas, or combinations thereof.
[39]
39. Lateral flow device, according to claim 30, characterized in that the infection is caused by a fungus.
[40]
40. Lateral flow device, according to claim 39, characterized in that the fungus is Aspergillus, Blastomyces, Candidiasis, Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp. Histoplasma sp. (as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), Stachybotrys (such as Stachybotrys chartarum), Mucroymcosis, Sporothrix, fungal eye infections, ringworm, Exserohilum, Cladosporium, Geotrichum, Saccharomyces, a species of Hansenula, a species of Candida, a species of Kluyveromyces, a species of Debaryomyces , a kind of Pichia, a kind of
Penicillium,y a species of Cladosporium, a species of Byssochlamys or a combination thereof.
[41]
41. Lateral flow device, according to claim 30, characterized in that the infection is caused by a protozoan.
[42]
42. Lateral flow device according to claim 41, characterized in that the protozoan is a Euglenozoa, a Heterolobosea, a Diplomonadida, an Amoebozoa, a Blastocystic, an Apicomplexa, or combinations thereof.
[43]
43. Lateral flow device, according to claim 30, characterized in that the infection is caused by a parasite.
[44]
44, Lateral flow device, according to claim 43, characterized in that the parasite is Trypanosoma cruzi (Chagas' disease), T. brucei gambiense, T. brucei rhodesiense, Leishmania braziliensis, L. infantum, L. mexicana , L. major, L. tropica, L. donovani, Naegleria fowleri, Giardia intestinalis (G. lamblia, G. duodenalis), canthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica, Blastocystic hominis, Babesia microti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and Toxoplasma gondii, or a combination thereof.
[45]
45. Lateral flow device, according to claim 1, characterized in that the sample is a biological sample or an environmental sample.
[46]
46. Lateral flow device, according to claim 45, characterized in that the biological sample is blood, plasma, serum, urine, feces, sputum, mucosa, lymphatic fluid, synovial fluid, bile, ascites, pleural effusion , seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., (e.g. a normal joint or a joint affected by diseases such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis) or a swab from the surface of the skin or mucous membrane.
[47]
47. Side flow device, according to claim 45, characterized in that the environmental sample is obtained from a food sample, paper surface, fabric, metal surface, wooden surface, plastic surface, sample of soil, freshwater sample, a wastewater sample, a saline water sample, or a combination thereof.
[48]
48. Lateral flow device according to claim 31, characterized in that the disease state is an infection, an organ disease, a blood disease, an immune system disease, a cancer, a brain disease and the nervous system, an endocrine disease, a disease related to pregnancy or childbirth, an inherited disease, or a disease acquired in the environment.
[49]
49. Lateral flow device, according to claim 26, characterized in that said disease state is characterized by the presence or absence of a gene or transcript or polypeptide of susceptibility or resistance to antibiotics or drugs, preferably in a pathogen or in a cell.
[50]
50. Lateral flow device, according to claim 49, characterized in that the one or more guide molecules identify a biological material.
[51]
51. Lateral flow device, according to claim 50, characterized in that the biological material is a genetically modified material.
[52]
52. Lateral flow device, according to claim 51, characterized in that the genetically modified material is a genetically modified plant.
[53]
53. Lateral flow device, according to claim 2, characterized in that the first molecule is FITC and the second molecule is FAM.
[54]
54. Lateral flow device, according to claim 35, characterized in that the viral infection is caused by the dengue virus.
[55]
55. A lateral flow device, characterized in that it comprises a substrate comprising a first end, wherein the first end comprises a sample loading portion and a first region loaded with a detectable binder, two or more CRISPR effector systems, two or more detection constructs, one or more first capture regions, each comprising a first binding agent, two or more second capture regions, each comprising a second binding agent, wherein each of the two or more effector systems CRISPR comprises a CRISPR effector protein and one or more guide sequences, each guide sequence configured to bind one or more target molecules.
[56]
56. Lateral flow device, according to claim 55, characterized in that each of the two or more detection constructs comprises — an RNA or DNA oligonucleotide, comprising a first molecule at a first end and a second molecule at a a second end.
[57]
57. Lateral flow device, according to claim 56, characterized in that it comprises two CRISPR effector systems and two detection constructs.
[58]
58. Lateral flow device, according to claim 56, characterized in that it comprises four CRISPR effector systems and four detection constructs.
[59]
59. A lateral flow device according to claim 55, characterized in that the sample loading portion further comprises one or more amplification reagents to amplify one or more target molecules.
[60]
60. A lateral flow device according to claim 57, characterized in that a first detection construct comprises FAM as a first molecule and biotin as a second molecule or vice versa and a second detection construct comprises FAM as a first molecule and Digoxigenin (DIG) as a second molecule or vice versa.
[61]
61. Lateral flow device, according to claim 60, characterized in that the CRISPR effector protein is an RNA targeting effector protein.
[62]
62. Lateral flow device, according to claim 61, characterized in that the RNA targeting effector protein is C2c2.
[63]
63. Lateral flow device, according to claim 19, characterized in that the RNA targeting effector protein is Casl3b.
[64]
64. A lateral flow device according to claim 58, characterized in that a first detection construct comprises Tye665 as a first molecule and Alexa-fluor-488 as a second molecule or vice versa; wherein a second detection construct comprises Tye665 as a first molecule and FAM as a second molecule or vice versa; wherein a third detection construct comprises Tye665 as a first molecule and biotin as a second molecule or vice versa; and wherein a fourth detection construct comprises Tye665 as a first molecule and DIG as a second molecule or vice versa.
[65]
65. Lateral flow device, according to claim 64, characterized in that the CRISPR effector protein is an RNA targeting or DNA targeting effector protein.
[66]
66. Lateral flow device, according to claim 65, characterized in that the RNA targeting effector is C2c2.
[67]
67. Lateral flow device, according to claim 65, characterized in that the RNA targeting effector is Casl3b.
[68]
68. Lateral flow device, according to claim 65, characterized in that the DNA targeting effector protein is Casl2a.
[69]
69. A method for detecting a target nucleic acid in a sample comprising contacting a sample with the first end of the lateral flow device as defined in any one of claims 1 to 53 comprising the sample loading portion; wherein the sample flows from the sample loading portion of the substrate to the first and second capture regions and generates a detectable signal.
[70]
70. Method according to claim 54, characterized in that the sample is a liquid sample or in which the sample has been dissolved in an aqueous solvent.
[71]
71. Method according to claim 54, characterized in that the sample does not contain target nucleic acid.
[72]
72. Method according to claim 56, characterized in that the detectable signal appears in the first capture region.
[73]
73. Method according to claim 54, characterized in that the sample contains target nucleic acid.
[74]
74. Method according to claim 58, characterized in that the detectable signal appears in the second capture region.
[75]
75. Method according to claims 58 or 59 characterized in that the presence of target nucleic acid is indicative of a disease state.
“ &. She: TIA Na. | E — Qf * La. "The 1 | 3
to you

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