Variant type V CRISPR/Cas effector polypeptides and methods of use thereof
The present disclosure provides variant type V CRISPR/Cas effector polypeptides, fusion polypeptides comprising the variant type V CRISPR/Cas effector polypeptides, and nucleic acids comprising nucleotide sequences encoding the variant polypeptides and fusion polypeptides. The present disclosure provides methods of binding, or binding and nicking, a target nucleic acid, using a variant type V CRISPR/Cas effector polypeptide of the present disclosure. The present disclosure provides methods of detecting a single-stranded DNA, using a variant type V CRISPR/Cas effector polypeptide of the present disclosure.
1. A variant CasX polypeptide comprising 85% or more identity to SEQ ID NO: 1 or 2, wherein amino acids 863-873 of SEQ ID NO: 1 or amino acids 850-860 of SEQ ID NO: 2 are replaced with a glycine polymer, a glycine-serine polymer, a glycine-alanine polymer, or an alanine-serine polymer, wherein the variant CasX polypeptide retains DNA binding activity and wherein the variant CasX polypeptide does not exhibit double-stranded DNA cleavage activity.
2. A method of binding, or binding and nicking, a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) the variant CasX polypeptide of claim 1 ; and
b) a CasX guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid.
3. 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 CasX fusion polypeptide comprising the variant CasX polypeptide of claim 1 fused to a heterologous polypeptide; and
b) a CasX guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid.
4. A method of detecting a target single-stranded DNA in a sample, the method comprising:
(a) contacting the sample with:
(i) the variant CasX polypeptide of claim 1 ;
(ii) a guide RNA comprising: a region that binds to the variant CasX polypeptide, and a guide sequence that hybridizes with the target DNA; and
(iii) a detector DNA that is single stranded and does not hybridize with the guide sequence of the guide RNA; and
(b) measuring a detectable signal produced by cleavage of the single-stranded detector DNA by the variant CasX polypeptide, thereby detecting the target DNA.
5. The variant CasX polypeptide of claim 1 , wherein amino acids 863-873 of SEQ ID NO:1 or amino acids 850-860 of SEQ ID NO:2 are replaced with the glycine-serine polymer.
6. The variant CasX polypeptide of claim 1 , wherein amino acids 863-873 of SEQ ID NO:1 or amino acids 850-860 of SEQ ID NO:2 are replaced with GGSGGSGGSG (SEQ ID NO: 100).
7. A CasX fusion polypeptide comprising:
a) the variant CasX polypeptide of claim 1 ; and
b) a heterologous polypeptide.
8. A method of binding, or binding and nicking, a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) the fusion polypeptide of claim 7 ; and
b) a CasX guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid.
9. The fusion polypeptide of claim 7 , wherein the fusion polypeptide comprises one or more nuclear localization sequences (NLSs).
10. The fusion polypeptide of claim 7 , wherein the heterologous polypeptide comprises a targeting polypeptide that provides for binding to a cell surface moiety on a target cell or target cell type.
11. The fusion polypeptide of claim 7 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies target DNA.
12. The fusion polypeptide of claim 7 , wherein the heterologous polypeptide exhibits one or more DNA-modifying enzymatic activities selected from: nuclease activity, methyltransferase activity, demethylase activity, deamination activity, depurination activity, integrase activity, transposase activity, and recombinase activity.
13. The fusion polypeptide of claim 7 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies a histone associated with a target nucleic acid.
14. The fusion polypeptide of claim 7 , wherein the heterologous polypeptide comprises a protein that increases or decreases transcription.
15. A nucleic acid comprising a nucleotide sequence encoding a variant CasX polypeptide comprising 85% or more identity to SEQ ID NO: 1 or 2, wherein amino acids 863-873 of SEQ ID NO: 1 or amino acids 850-860 of SEQ ID NO: 2 are replaced with a glycine polymer, a glycine-serine polymer, a glycine-alanine polymer, or an alanine-serine polymer, wherein the variant CasX polypeptide retains DNA binding activity and wherein the variant CasX polypeptide does not exhibit double-stranded DNA cleavage activity.
16. A recombinant expression vector comprising the nucleic acid of claim 15 .
17. An in vitro cell comprising the nucleic acid of claim 15 .
18. The cell of claim 17 , wherein the cell is a eukaryotic cell.
19. The nucleic acid of claim 15 , further comprising a nucleotide sequence encoding a CasX guide RNA.
20. A nucleic acid comprising a nucleotide sequence encoding a CasX fusion polypeptide, the CasX fusion polypeptide comprising:
a) a variant CasX polypeptide comprising 85% or more identity to SEQ ID NO: 1 or 2, wherein amino acids 863-873 of SEQ ID NO: 1 or amino acids 850-860 of SEQ ID NO: 2 are replaced with a glycine polymer, a glycine-serine polymer, a glycine-alanine polymer, or an alanine-serine polymer, wherein the variant CasX polypeptide retains DNA binding activity and wherein the variant CasX polypeptide does not exhibit double-stranded DNA cleavage activity; and
b) a heterologous polypeptide.
21. The nucleic acid of claim 20 , further comprising a nucleotide sequence encoding a CasX guide RNA.
22. A composition comprising:
a) the variant CasX polypeptide of claim 1 , or the nucleic acid of claim 15 ; and
b) a CasX guide RNA, or one or more DNA molecules comprising a nucleotide sequence encoding the CasX guide RNA.
23. A composition comprising:
a) the fusion polypeptide of claim 7 , or the nucleic acid of claim 20 ; and
b) a CasX guide RNA, or one or more DNA molecules comprising a nucleotide sequence encoding the CasX guide RNA.