GB2622995A - CAS13-based compositions and methods of use thereof - Google Patents
CAS13-based compositions and methods of use thereof Download PDFInfo
- Publication number
- GB2622995A GB2622995A GB2319843.5A GB202319843A GB2622995A GB 2622995 A GB2622995 A GB 2622995A GB 202319843 A GB202319843 A GB 202319843A GB 2622995 A GB2622995 A GB 2622995A
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- United Kingdom
- Prior art keywords
- sequence
- seq
- rna
- cas13
- composition
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- Pending
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- 238000000034 method Methods 0.000 title claims 14
- 239000000203 mixture Substances 0.000 title claims 14
- 101100385364 Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CCM 3970 / CIP 100100 / NCTC 11856 / SLCC 3954 / 1120) cas13 gene Proteins 0.000 title 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 26
- 108090000623 proteins and genes Proteins 0.000 claims 21
- 102000004169 proteins and genes Human genes 0.000 claims 21
- 239000012636 effector Substances 0.000 claims 14
- 239000002773 nucleotide Substances 0.000 claims 8
- 125000003729 nucleotide group Chemical group 0.000 claims 8
- 108091033319 polynucleotide Proteins 0.000 claims 7
- 102000040430 polynucleotide Human genes 0.000 claims 7
- 239000002157 polynucleotide Substances 0.000 claims 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 6
- 239000013598 vector Substances 0.000 claims 6
- 238000010453 CRISPR/Cas method Methods 0.000 claims 4
- 108091034117 Oligonucleotide Proteins 0.000 claims 4
- 210000004027 cell Anatomy 0.000 claims 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims 4
- 210000001236 prokaryotic cell Anatomy 0.000 claims 4
- 102100029791 Double-stranded RNA-specific adenosine deaminase Human genes 0.000 claims 3
- 101000865408 Homo sapiens Double-stranded RNA-specific adenosine deaminase Proteins 0.000 claims 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 238000003776 cleavage reaction Methods 0.000 claims 3
- 239000003550 marker Substances 0.000 claims 3
- 230000007017 scission Effects 0.000 claims 3
- 108020004414 DNA Proteins 0.000 claims 2
- 102000053602 DNA Human genes 0.000 claims 2
- 108010021843 fluorescent protein 583 Proteins 0.000 claims 2
- 125000006850 spacer group Chemical group 0.000 claims 2
- 230000035897 transcription Effects 0.000 claims 2
- 238000013518 transcription Methods 0.000 claims 2
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 claims 1
- 102000055025 Adenosine deaminases Human genes 0.000 claims 1
- 241001135756 Alphaproteobacteria Species 0.000 claims 1
- 241000894006 Bacteria Species 0.000 claims 1
- 108020004705 Codon Proteins 0.000 claims 1
- 102100038191 Double-stranded RNA-specific editase 1 Human genes 0.000 claims 1
- 102100024692 Double-stranded RNA-specific editase B2 Human genes 0.000 claims 1
- 206010059866 Drug resistance Diseases 0.000 claims 1
- 241000206602 Eukaryota Species 0.000 claims 1
- 101000742223 Homo sapiens Double-stranded RNA-specific editase 1 Proteins 0.000 claims 1
- 101000686486 Homo sapiens Double-stranded RNA-specific editase B2 Proteins 0.000 claims 1
- 108091007460 Long intergenic noncoding RNA Proteins 0.000 claims 1
- 108091005804 Peptidases Proteins 0.000 claims 1
- 239000004365 Protease Substances 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 102000004389 Ribonucleoproteins Human genes 0.000 claims 1
- 108010081734 Ribonucleoproteins Proteins 0.000 claims 1
- 108700005077 Viral Genes Proteins 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 claims 1
- 238000010367 cloning Methods 0.000 claims 1
- 230000000536 complexating effect Effects 0.000 claims 1
- 230000001419 dependent effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 238000011901 isothermal amplification Methods 0.000 claims 1
- 230000004807 localization Effects 0.000 claims 1
- 108020004999 messenger RNA Proteins 0.000 claims 1
- 108020004707 nucleic acids Proteins 0.000 claims 1
- 102000039446 nucleic acids Human genes 0.000 claims 1
- 150000007523 nucleic acids Chemical class 0.000 claims 1
- 239000013612 plasmid Substances 0.000 claims 1
- 238000010839 reverse transcription Methods 0.000 claims 1
- 241001515965 unidentified phage Species 0.000 claims 1
- 239000013603 viral vector Substances 0.000 claims 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cosmetics (AREA)
Abstract
Claims (1)
- CLAIMS We claim: 1. A polynucleotide comprising a nucleotide sequence encoding a class II, type VI CRISPR/Cas effector protein (Cas13) and optionally a heterologous sequence, wherein the Cas effector protein comprises an amino acid sequence encoded by SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67, or a sequence with at least 70% sequence identity thereto; optionally wherein the sequence encoding the Cas effector protein comprises SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67, or a sequence with at least 70% sequence identity to SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67. 2. The polynucleotide of claim 1, wherein the sequence encoding the Cas effector protein is codon optimized for expression in a prokaryotic or eukaryotic cell. 3. The polynucleotide of claim 1 or 2, comprising a heterologous sequence, wherein the heterologous sequence comprises a promoter, transcription terminator, multiple cloning site, drug resistance marker, one or more protease recognition sites, one or more epitope tags, or a combination thereof. 4. The polynucleotide of claim 3, wherein the heterologous sequence is operably linked to the sequence encoding the Cas effector protein. 5. The polynucleotide of any one of claims 1-4 further comprising a sequence encoding an RNA comprising a crRNA sequence, wherein the RNA is capable of complexing with the Cas effector protein and hybridizing to a target RNA sequence. 6. The polynucleotide of any one of claims 1-5, wherein the Cas effector protein is derived from Thermoclostridium caenicola or a Proteobacteria bacterium. 7. A vector comprising the polynucleotide of any one of claims 1-6, optionally wherein the vector comprises the nucleotide sequence of SEQ ID 184 45497470v11. NO:69 or SEQ ID NO:70, or a sequence having at least 60% sequence identity to SEQ ID NO:69 or SEQ ID NO:70. 8. The vector of claim 7, wherein the vector is a viral vector or plasmid. 9. A prokaryotic or eukaryotic cell comprising the vector of claim 7 or 8. 10. A method of producing a class II, type VI CRISPR/Cas effector protein comprising contacting the vector of claim 7 or 8 with a prokaryotic or eukaryotic cell under conditions suitable for expression of the sequence encoding the Cas effector protein. 11. The method of claim 10 further comprising isolating and/or purifying the Cas effector protein. 12. An isolated class II, type VI CRISPR/Cas effector protein, wherein the Cas effector protein is produced by the method of claim 10 or 11. 13. An isolated class II, type VI CRISPR/Cas effector protein comprising the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:66, or a sequence with at least 70% sequence identity to SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68. 14. A ribonucleoprotein complex comprising the Cas effector protein of claim 12 or 13 complexed with an RNA comprising crRNA sequence, optionally wherein the RNA is capable of hybridizing to a target RNA sequence. 15. A composition comprising a Cas13 protein, wherein the Cas13 protein comprises the amino acid sequence of SEQ ID NO:64, SEQ ID NO:66 or SEQ ID NO:68, or a sequence with at least 70% sequence identity to SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68. 16. The composition of claim 15, wherein the Cas13 protein comprises one or more HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains, preferably two HEPN domains. 17. The composition of claim 16, wherein the HEPN domain comprises a RxxxxH (SEQ ID NO:192) motif sequence, wherein X represents any amino acid. 185 45497470v118. The composition of any one of claims 15-17, wherein the Cas13 protein is complexed with and RNA comprising a crRNA sequence, optionally wherein the RNA is capable of hybridizing to a target RNA sequence19. The composition of claim 18, wherein the crRNA sequence comprises a spacer sequence that is capable of hybridizing to the target RNA sequence, and a direct repeat sequence20. The composition of claim 18 or 19, wherein the crRNA sequence comprises a spacer of about 20-30 nucleotides, preferably 24-28 nucleotides21. The composition of any one of claims 18-20, wherein the Cas13 protein can cleave the target RNA sequence at a temperature of about 37-70 oC, about 50-70 oC, about 47-60 oC, or about 60 ºC22. The composition of any one of claims 18-20, wherein the Cas13 protein can cleave the target RNA sequence at a temperature of about 37 oC- 42 °C, preferably about 37 oC23. The composition of any one of claims 18-22 comprised in a prokaryotic or eukaryotic cell24. A method of performing targeted knockdown of an RNA transcript comprising introducing the composition of any one of claims 18-22 to a cell, wherein the crRNA sequence hybridizes to the RNA transcript, thereby inducing cleavage of the RNA transcript by the Cas13 protein25. The method of claim 24, wherein RNA transcript is a mRNA or lincRNA26. The method of claim 24, wherein RNA transcript is derived from a viral gene, preferably a bacteriophage .27. A method of detecting the presence of an RNA transcript in a nucleic acid sample comprising contacting the sample with the composition of any one of claims 18-22 in the presence of an activatable single stranded RNA (ssRNA) oligonucleotide comprising a reporter moiety, wherein the crRNA is designed to hybridize to the RNA transcript, wherein the Cas13 cleaves the ssRNA oligonucleotide upon binding of the 186 45497470v1 Cas13 crRNA complex to the RNA transcript, wherein detection of the cleavage of the ssRNA oligonucleotide indicates the presence of the RNA transcript.28. The method of claim 27, wherein the RNA transcript is generated by transcription from a dsDNA molecule, optionally wherein the dsDNA molecule is generated by reverse transcription coupled isothermal amplification of a target RNA. 29. The method of claim 27 or 28, wherein the reporter moiety comprises a fluorophore linked to a quencher via the ssRNA, wherein fluorescence is emitted upon cleavage of the ssRNA oligonucleotide. 30. A method of determining the localization of an RNA transcript in a cell comprising introducing the composition of any one of claims 18-22 to the cell, wherein the Cas13 is catalytically inactive and further comprises a detectable marker, wherein the crRNA is designed to hybridize to the RNA transcript, and wherein the Cas13 crRNA complex binds to the RNA transcript, thereby indicating the location of the RNA transcript. 31. The method of claim 30, wherein the detectable marker is selected from GFP, eGFP, RFP, YFP, BFP, CFP, mNeonGreen, mCherry, mOrange, mRaspberry, mPlum, mKO, mRFP, mRFPruby, mRuby, tagRFP, mKate2, and DsRed. 32. A method for performing targeted editing of an RNA transcript comprising introducing the composition of any one of claims 18-22 to a cell, wherein the Cas13 is catalytically inactive and further comprises a deaminase domain of an RNA-dependent Adenosine Deaminase (ADAR), wherein the crRNA is capable of hybridizing with a region in the RNA transcript comprising a target A nucleotide to form an RNA duplex, wherein the duplex comprises an A-C mismatch at the target A nucleotide, wherein the target A nucleotide is deaminated by the deaminase domain. 187 45497470v133. The method of claim 32, wherein the ADAR is selected from ADAR1, ADAR2, or ADAR3. 188 45497470v1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202163194099P | 2021-05-27 | 2021-05-27 | |
| PCT/IB2021/054664 WO2021240443A1 (en) | 2020-05-27 | 2021-05-27 | Iscan: an rt-lamp-coupled crispr-cas module for rapid, sensitive detection of sars-cov-2 |
| PCT/IB2022/055036 WO2022249152A2 (en) | 2021-05-27 | 2022-05-27 | Cas13-based compositions and methods of use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB202319843D0 GB202319843D0 (en) | 2024-02-07 |
| GB2622995A true GB2622995A (en) | 2024-04-03 |
Family
ID=82016483
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB2319843.5A Pending GB2622995A (en) | 2021-05-27 | 2022-05-27 | CAS13-based compositions and methods of use thereof |
Country Status (2)
| Country | Link |
|---|---|
| GB (1) | GB2622995A (en) |
| WO (1) | WO2022249152A2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117701530A (en) * | 2022-12-08 | 2024-03-15 | 广州瑞风生物科技有限公司 | Cas protein truncate, method for constructing same and application thereof |
| CN117106101B (en) * | 2023-10-20 | 2024-06-07 | 合肥百裕生物科技有限公司 | Plasmid and ASFV protease inhibitor screening and drug effect evaluation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3692145A4 (en) | 2017-10-04 | 2021-11-24 | The Broad Institute, Inc. | Systems, methods, and compositions for targeted nucleic acid editing |
-
2022
- 2022-05-27 WO PCT/IB2022/055036 patent/WO2022249152A2/en active Application Filing
- 2022-05-27 GB GB2319843.5A patent/GB2622995A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| Not Advised * |
Also Published As
| Publication number | Publication date |
|---|---|
| GB202319843D0 (en) | 2024-02-07 |
| WO2022249152A2 (en) | 2022-12-01 |
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