RNA-guided nucleic acid modifying enzymes and methods of use thereof
The present disclosure provides CasY proteins, nucleic acids encoding the CasY proteins, and modified host cells comprising the CasY proteins and/or nucleic acids encoding same. CasY proteins are useful in a variety of applications, which are provided. The present disclosure provides CasY guide RNAs that bind to and provide sequence specificity to the CasY proteins, nucleic acids encoding the CasY guide RNAs, and modified host cells comprising the CasY guide RNAs and/or nucleic acids encoding same. CasY guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides methods of identifying a CRISPR RNA-guided endonuclease.
1. A composition comprising:
a) a CasY fusion polypeptide, or a nucleic acid molecule encoding the CasY fusion polypeptide, wherein the CasY fusion polypeptide comprises a CasY polypeptide fused to a heterologous polypeptide, and wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2; and
b) a CasY guide RNA, or one or more DNA molecules encoding the CasY guide RNA.
2. The composition of claim 1 , wherein the CasY polypeptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
3. The composition of claim 1 , wherein the heterologous polypeptide is a nuclear localization signal.
4. The composition of claim 1 , comprising one or more of: a buffer, a nuclease inhibitor, a lipid, and a protease inhibitor.
5. The composition of claim 1 , wherein the CasY polypeptide is a catalytically inactive CasY Polypeptide (dCasY).
6. The composition of claim 5 , wherein the CasY polypeptide comprises one or more mutations at a position corresponding to those selected from: D828, E914, and D1074 of SEQ ID NO: 1.
7. The composition of claim 1 , further comprising a DNA donor template.
8. A CasY fusion polypeptide comprising: a CasY polypeptide fused to a heterologous polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
9. The CasY fusion polypeptide of claim 8 , wherein the CasY polypeptide comprises an amino acid sequence having 59% 80% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
10. The CasY fusion polypeptide of claim 8 , wherein the CasY polypeptide is a catalytically inactive CasY Polypeptide (dCasY).
11. The CasY fusion polypeptide of claim 10 , wherein the CasY polypeptide comprises one or more mutations at a position corresponding to those selected from: D828, E914, and D1074 of SEQ ID NO: 1.
12. The CasY fusion polypeptide of claim 8 , wherein the heterologous polypeptide is a targeting polypeptide that provides for binding to a cell surface moiety on a target cell or target cell type.
13. The CasY fusion polypeptide of claim 8 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies target DNA.
14. The CasY fusion polypeptide of claim 8 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies a target polypeptide associated with a target nucleic acid.
15. The CasY fusion polypeptide of claim 8 , wherein the heterologous polypeptide is an endosomal escape polypeptide.
16. The CasY fusion polypeptide of claim 8 , wherein the heterologous polypeptide is a protein that increases or decreases transcription.
17. A nucleic acid comprising a nucleotide sequence encoding a CasY fusion polypeptide, wherein the CasY fusion polypeptide comprises a CasY polypeptide fused to a heterologous polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
18. One or more nucleic acid molecules encoding:
(a) a CasY guide RNA; and
(b) a CasY fusion polypeptide, wherein the CasY fusion polypeptide comprises a CasY polypeptide fused to a heterologous polypeptide, and wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
19. The one or more nucleic acid molecules of claim 18 , wherein the CasY polypeptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
20. A eukaryotic cell comprising one or more of:
a) a CasY polypeptide, or a nucleic acid molecule encoding the CasY polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2;
b) a CasY fusion polypeptide, or a nucleic acid molecule encoding the CasY fusion polypeptide, wherein the CasY fusion polypeptide comprises a CasY polypeptide fused to a heterologous polypeptide, and wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2; and
c) a CasY guide RNA, or a nucleic acid molecule encoding the CasY guide RNA.
21. A cell comprising a CasY fusion polypeptide, or a nucleic acid molecule encoding the CasY fusion polypeptide, wherein the CasY fusion polypeptide comprises a CasY polypeptide fused to a heterologous polypeptide, and wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
22. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) a CasY polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2; and
b) a CasY guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid,
wherein the target nucleic acid is double-stranded DNA, and
wherein said contacting results in modification of the target nucleic acid by the CasY polypeptide.
23. The method of claim 22 , wherein said modification is cleavage of the target nucleic acid.
24. The method of claim 22 , wherein the target nucleic acid is genomic DNA or extrachromosomal DNA.
25. The method of claim 22 , wherein said contacting takes place in vitro outside of a cell.
26. The method of claim 22 , wherein said contacting takes place inside of a cell in vivo.
27. The method of claim 26 , wherein the cell is a eukaryotic cell.
28. A method of modulating transcription from a target DNA, modifying a target nucleic acid, or modifying a protein associated with a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) a CasY fusion polypeptide comprising a CasY polypeptide fused to a heterologous polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEO ID NO: 1 or SEO ID NO:2; and
b) a CasY guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid,
wherein the target nucleic acid is double-stranded DNA.