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 method of editing a target nucleotide sequence in a target DNA, the method comprising contacting the target nucleotide sequence with:
a) a fusion polypeptide comprising:
i) an enzymatically active RNA-guided endonuclease, wherein the endonuclease is a nickase that introduces a single-stranded break in a target DNA; and
ii) a DNA polymerase, wherein the DNA polymerase synthesizes a new strand on the target DNA,
wherein the fusion polypeptide does not include a deaminase; and
b) a guide RNA that comprises:
i) a protein-binding segment comprising a nucleotide sequence that binds to the RNA-guided endonuclease; and
ii) a target-binding segment comprising a nucleotide sequence that is complementary to a target nucleotide sequence in a target nucleic acid,
wherein the DNA polymerase binds to the target DNA 5′ to the single-stranded break, and synthesizes a new strand on the target DNA.
2. The method of claim 1 , wherein the endonuclease comprises a class 2 CRISPR/Cas endonuclease.
3. The method of claim 2 , wherein the class 2 CRISPR/Cas endonuclease comprises a type V or type VI CRISPR/Cas endonuclease.
4. The method of claim 2 , wherein the class 2 CRISPR/Cas endonuclease comprises a Cas9 polypeptide.
5. The method of claim 1 , wherein the fusion polypeptide comprises a linker connecting the endonuclease and the DNA polymerase.
6. The method of claim 1 , wherein the fusion polypeptide comprises a nuclear localization signal.
7. The method of claim 3 , wherein the new strand comprises a mutation.
8. The method of claim 3 , wherein the new strand comprises a correction of a mutation.
9. The method of claim 1 , wherein the target nucleic acid is present in a eukaryotic cell.
10. The method of claim 9 , wherein the cell is in vivo.
11. The method of claim 9 , wherein the endonuclease, the DNA polymerase, and the guide RNA are encoded by one or more nucleic acids that are introduced into the cell.
12. The method of claim 9 , wherein the endonuclease, the DNA polymerase, and the guide RNA are exogenously introduced into the cell.