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 method of cleaving double stranded DNA (dsDNA) in a cell, the method comprising contacting the cell with:
a) a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 109-126; and
b) a guide nucleic acid, wherein the guide nucleic acid comprises:
i) a repeat sequence; and
ii) a spacer sequence, wherein the spacer sequence hybridizes to a target sequence on a target strand of the dsDNA, wherein:
the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5′-NTTN-3′ and the PAM is located 5′ of the target sequence on the non-target strand, and
the polypeptide and the guide nucleic acid form a ribonucleoprotein (RNP) complex that binds the target sequence and cleaves the dsDNA.
2. The method of claim 1 , wherein the RNP complex cleaves both strands of the dsDNA.
3. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to any one the amino acid sequences depicted in any one of SEQ ID NOs: 109-126.
4. The method of claim 1 , wherein the nucleic acid encoding the polypeptide and the guide nucleic acid are contained in a lipid nanoparticle (LNP), and the method comprises contacting the cell with the LNP.
5. The method of claim 1 , wherein the cell is a human cell.
6. The method of claim 1 , wherein the length of the spacer sequence is 14 to 24 nucleotides.
7. The method of claim 1 , wherein the nucleic acid encoding the polypeptide comprises a messenger RNA.
8. The method of claim 1 , comprising contacting the cell with a donor nucleic acid.
9. The method of claim 8 , wherein the donor nucleic acid is present in an adeno-associated viral (AAV) vector.
10. The method of claim 1 , wherein the cell is a liver cell.
11. The method of claim 1 , wherein the cell is a blood cell.
12. The method of claim 1 , wherein the guide nucleic acid comprises a 2′-O-Methyl modified nucleotide, a phosphorothioate linkage, or a combination thereof.
13. The method of claim 1 , wherein the nucleic acid encoding the polypeptide encodes a nuclear localization signal (NLS), wherein the polypeptide is fused to the NLS upon expression of the nucleic acid in the cell.
14. The method of claim 1 , wherein the method removes at least one nucleotide from the target sequence.
15. A composition for cleaving double stranded DNA (dsDNA) in a cell, the composition comprising:
a) a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 109-126; and
b) a guide nucleic acid, wherein the guide nucleic acid comprises:
i) a repeat sequence; and
ii) a spacer sequence, wherein the spacer sequence hybridizes to a target sequence on a target strand of the dsDNA, wherein:
the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5′-NTTN-3′ and the PAM is located 5′ of the target sequence on the non-target strand, and
the polypeptide and the guide nucleic acid form a ribonucleoprotein (RNP) complex that binds the target sequence and cleaves the dsDNA.
16. A pharmaceutical composition comprising the composition of claim 15 and a pharmaceutically acceptable excipient.
17. The method of claim 1 , wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 120.
18. The method of claim 17 , wherein the repeat sequence comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 181.
19. The composition of claim 15 , comprising an LNP, wherein the nucleic acid encoding the polypeptide and the guide nucleic acid are contained in the LNP.
20. The composition of claim 15 , wherein the nucleic acid encoding the polypeptide comprises a messenger RNA.
21. The composition of claim 15 , wherein the guide nucleic acid comprises a 2′-O-Methyl modified nucleotide, a phosphorothioate linkage, or a combination thereof.
22. The composition of claim 15 , wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 120.
23. The composition of claim 15 , wherein the repeat sequence comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 181.