CRISPR-associated (Cas) protein
A new CRISPR-associated (Cas) protein, termed “CasM,” is described, as well as polynucleotides encoding the same and methods of using CasM for site-specific genome engineering. CasM proteins are capable of targeting and cleaving single-stranded RNA.
1. A method of directing a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) nucleoprotein complex to a selected nucleic acid target sequence, the method comprising:
contacting the selected nucleic acid target sequence with one or more nucleoprotein complexes comprising
a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein, wherein the Cas protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:44, an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:37, an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:39, and an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:44; and
a cognate nucleic acid guide comprising a repeat sequence and a spacer sequence, wherein the repeat sequence and the spacer sequence do not naturally occur together, wherein the Cas protein and the cognate nucleic acid guide form the nucleoprotein complex, and wherein the nucleoprotein complex binds to the selected nucleic acid target sequence.
2. The method of claim 1 , wherein the nucleic acid target sequence comprises RNA.
3. The method of claim 1 , wherein the method is performed in a cell.
4. The method of claim 3 , wherein the cell is a eukaryotic cell.
5. The method of claim 3 , wherein the cell constitutively expresses the Cas protein.
6. The method of claim 1 , wherein the Cas protein is fused to a protein.
7. The method of claim 6 , wherein the protein which is fused to the Cas protein comprises an enzyme.
8. The method of claim 1 , wherein the cognate nucleic acid guide comprises RNA.
9. The method of claim 1 , wherein the cognate nucleic acid guide comprises DNA.
10. The method of claim 1 , wherein the Cas protein comprises an amino acid sequence having at least 98 percent sequence identity to SEQ ID NO:44.
11. The method of claim 1 , wherein the nucleoprotein complex is capable of site-directed binding to the nucleic acid target sequence.
12. The method of claim 11 , wherein the Cas protein is catalytically active, resulting in the binding of the nucleoprotein complex to the nucleic acid target sequence and cleaving of the nucleic acid target sequence.
13. The method of claim 12 , whereby the cleaving results in one or more single-strand or double-strand breaks in the nucleic acid target sequence.
14. The method of claim 12 , wherein the nucleic acid target sequence comprises RNA.
15. The method of claim 12 , wherein the method is performed in a cell.
16. The method of claim 15 , wherein the cell constitutively expresses the Cas protein.
17. The method of claim 15 , wherein the cell comprises a eukaryotic cell.
18. The method of claim 17 , wherein the eukaryotic cell comprises a mammalian cell.
19. The method of claim 18 , wherein the mammalian cell comprises a human cell.
20. The method of claim 19 , wherein the human cell comprises a stem cell.
21. A method of directing a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) nucleoprotein complex to a selected nucleic acid target sequence, the method comprising:
contacting the selected nucleic acid target sequence with one or more nucleoprotein complexes comprising
a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein, wherein the Cas protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:39, and SEQ ID NO:44; and
a cognate nucleic acid guide comprising a repeat sequence and a spacer sequence, wherein the repeat sequence and the spacer sequence do not naturally occur together, wherein the Cas protein and the cognate nucleic acid guide form the nucleoprotein complex, and wherein the nucleoprotein complex binds to the selected nucleic acid target sequence.
22. The method of claim 21 , wherein the Cas protein comprises the amino acid sequence of SEQ ID NO: 37.
23. The method of claim 21 , wherein the Cas protein comprises the amino acid sequence of SEQ ID NO: 39.
24. The method of claim 21 , wherein the Cas protein comprises the amino acid sequence of SEQ ID NO: 44.
25. The method of claim 1 , wherein the Cas protein is catalytically inactive.