CRISPR-Cas effector polypeptides and methods of use thereof
The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.
1. A non-naturally occurring composition comprising:
a) a polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the polypeptide, wherein the amino acid sequence of the polypeptide is at least 95% identical to SEQ ID NO:120; and
b) a guide RNA comprising:
i) a first region comprising a guide sequence that hybridizes to a target sequence; and
ii) a second region that binds the polypeptide,
wherein the first region is heterologous to the second region and wherein the second region is 5′ to the first region; and
c) a target double-stranded DNA comprising:
i) the target sequence; and
ii) a protospacer adjacent motif (PAM) of 5′-NTTN-3′ wherein T is thymine and N is any nucleotide, wherein said PAM is immediately adjacent to the target sequence and wherein the PAM is on the non-target strand of the target double-stranded DNA, 5′ of the target sequence.
2. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIK” (amino acids 1-3 of SEQ ID NO: 120).
3. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIKP” (amino acids 1-4 of SEQ ID NO: 120).
4. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIKPT” (amino acids 1-5 of SEQ ID NO: 120).
5. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIKPTV” (amino acids 1-6 of SEQ ID NO: 120).
6. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIKPTVS” (amino acids 1-7 of SEQ ID NO: 120).
7. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIKPTVSQ” (amino acids 1-8 of SEQ ID NO: 120).
8. The composition of claim 1 , wherein the N terminus of the polypeptide begins with the amino acid sequence “MIKPTVSQF” (amino acids 1-9 of SEQ ID NO: 120).
9. The composition of claim 1 , wherein the recombinant guide RNA comprises a nucleobase sequence that is at least 80% identical to SEQ ID NO:181.
10. The composition of claim 1 , wherein the recombinant guide RNA comprises a nucleobase sequence that is at least 95% identical to SEQ ID NO:181.
11. The composition of claim 1 , comprising a nuclear localization signal (NLS) that is fused to the N terminus of the polypeptide, the C terminus of the polypeptide, or both termini of the polypeptide.
12. The composition of claim 11 , wherein the NLS is fused directly to the N-terminus of the polypeptide via an amide bond.
13. The composition of claim 1 , wherein the polypeptide is a nickase.
14. The composition of claim 1 , comprising a DNA donor template, an additional recombinant guide RNA.
15. The composition of claim 1 , comprising a lipid, a liposome, a vector, or a particle.
16. The composition of claim 1 , comprising one or more of: a buffer, a nuclease inhibitor, and a protease inhibitor.
17. A pharmaceutical composition comprising the composition of claim 1 , and a pharmaceutically acceptable excipient.
18. The composition of claim 1 , wherein the PAM comprises the sequence 5′-VTTN-3′, wherein V is selected from adenine, guanine, and cytosine and T is thymine.
19. The composition of claim 1 , comprising a heterologous polypeptide fused directly to the N terminus or C terminus of the polypeptide.
20. The composition of claim 1 , wherein the amino acid sequence of the polypeptide is at least 98% identical to SEQ ID NO: 120.
21. The composition of claim 1 , comprising a divalent metal ion.
22. The composition of claim 1 , comprising a magnesium cation or a salt thereof.
23. The composition of claim 1 , wherein the polypeptide comprises a RuvC active site capable of both cleaving DNA and binding a crRNA.
24. The composition of claim 1 , wherein the amino acid sequence of the polypeptide is 100% identical to SEQ ID NO:120.
25. The composition of claim 1 , wherein the polypeptide is a nuclease.
26. The composition of claim 1 , wherein a nuclease domain of the polypeptide is catalytically inactive or has reduced catalytic activity compared to a polypeptide that is 100% identical to SEQ ID NO:120.
27. The composition of claim 1 , comprising an additional guide RNA.
28. The composition of claim 1 , comprising a lipid nanoparticle.
29. The composition of claim 1 , comprising an adeno-associated viral (AAV) vector that comprises the nucleic acid encoding the polypeptide.
30. The composition of claim 29 , wherein the AAV vector encodes the guide RNA.