RNA-guided endonuclease fusion polypeptides and methods of use thereof
View Patent ↗The present disclosure provides a fusion polypeptide comprising: a) an enzymatically active RNA-guided endonuclease that introduces a single-stranded break in a target DNA; and b) an error-prone DNA polymerase. The present disclosure provides a system comprising: a) a fusion polypeptide of the present disclosure; and b) a guide RNA. The present disclosure provides a cell comprising a fusion polypeptide of the present disclosure, or a system of the present disclosure. The present disclosure provides a method of mutagenizing a target polynucleotide.
1. A fusion polypeptide comprising:
a) an enzymatically active RNA-guided endonuclease, wherein the endonuclease is a nickase that introduces a single-stranded break in a target DNA; and
b) a DNA polymerase, wherein the DNA polymerase synthesizes a new strand on the target DNA,
wherein the fusion polypeptide does not comprise a deaminase.
2. The fusion polypeptide of claim 1 , wherein the DNA polymerase introduces a mutation in the new strand.
3. The fusion polypeptide of claim 1 , wherein the fusion polypeptide, when complexes with a guide RNA, introduces a mutation in the new strand at a distance of from 1 nucleotide to 104 nucleotides from the single-stranded break in the target DNA.
4. The fusion polypeptide of claim 1 , wherein the endonuclease comprises a class 2 CRISPR/Cas endonuclease.
5. The fusion polypeptide of claim 4 , wherein the class 2 CRISPR/Cas endonuclease comprises a type V or type VI CRISPR/Cas endonuclease.
6. The fusion polypeptide of claim 4 , wherein the class 2 CRISPR/Cas endonuclease comprises a Cas9 polypeptide.
7. The fusion polypeptide of claim 1 , comprising a linker connecting the endonuclease and the DNA polymerase.
8. The fusion polypeptide of claim 1 , further comprising a nuclear localization signal.
9. The fusion polypeptide of claim 1 , wherein the fusion polypeptide, when complexed with a guide RNA, exhibits a target mutation rate of from 10 −8 to 10 −7 , 10 −7 to 10 −6 , 10 −6 to 10 −5 , 10 −5 to 10 −4 , or 10 −4 to 10 −3 mutations per nucleotide per genome replication event.
10. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) the enzymatically active RNA-guided endonuclease; and
b) the DNA polymerase.
11. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) the enzymatically active RNA-guided endonuclease;
b) a linker; and
c) the DNA polymerase.
12. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) a nuclear localization signal;
b) the enzymatically active RNA-guided endonuclease; and
c) the DNA polymerase.
13. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) a nuclear localization signal;
b) the enzymatically active RNA-guided endonuclease;
c) a linker; and
d) the DNA polymerase.
14. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) the DNA polymerase; and
b) the enzymatically active RNA-guided endonuclease.
15. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) the DNA polymerase;
b) a linker; and
c) the enzymatically active RNA-guided endonuclease.
16. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) a nuclear localization signal;
b) the DNA polymerase; and
c) the enzymatically active RNA-guided endonuclease.
17. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) a nuclear localization signal;
b) the DNA polymerase;
c) a linker; and
d) the enzymatically active RNA-guided endonuclease.
18. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of claim 1 .
19. A recombinant expression vector comprising the nucleic acid of claim 18 .
20. An editing method comprising:
contacting a target DNA with:
a) the fusion polypeptide of claim 1 ; and
b) a guide RNA comprising a nucleotide sequence that comprises a protein-binding segment comprising a nucleotide sequence that binds to the endonuclease, and a target-binding segment comprising a nucleotide sequence that is complementary to a target nucleotide sequence of the target DNA,
wherein the DNA polymerase binds to the target DNA 5′ to the single-stranded break, and synthesizes a new strand on the target DNA.
21. The method of claim 20 , wherein the new strand comprises a mutation.
22. The method of claim 20 , wherein the endonuclease comprises a class 2 CRISPR/Cas endonuclease.
23. The method of claim 22 , wherein the class 2 CRISPR/Cas endonuclease comprises a Cas9 polypeptide.
24. The method of claim 20 , wherein contacting the target DNA with the fusion polypeptide comprises introducing a nucleic acid encoding the fusion polypeptide into a cell comprising the target DNA.