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 . An editing system comprising:
a) an enzymatically active RNA-guided endonuclease that introduces a break in at least one strand in a target DNA;
b) a DNA polymerase that binds to the target DNA 5′ to the break; and
c) a guide RNA that binds the endonuclease,
wherein the endonuclease, DNA polymerase, and guide RNA are exogenously introduced into a cell, or are encoded by one or more exogenously introduced nucleic acids, wherein the endonuclease and the DNA polymerase are fused as parts of a fusion polypeptide, and
wherein the fusion polypeptide does not comprise a deaminase.
2 . The system of claim 1 , wherein the guide RNA 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.
3 . The system of claim 1 , wherein the guide RNA comprises one or more of:
a) a modified base;
b) a modified backbone;
c) a modified sugar moiety;
d) a non-natural internucleoside linkage.
4 . The system of claim 1 , wherein the guide RNA guides the endonuclease to introduce the break in the target DNA.
5 . The system of claim 1 , wherein the break in at least one strand is a single-stranded break.
6 . The system of claim 1 , wherein the DNA polymerase synthesizes a new strand on the target DNA.
7 . The system of claim 6 , wherein the DNA polymerase introduces a mutation in the new strand.
8 . The system of claim 7 , wherein the DNA polymerase introduces a mutation in the new strand at a distance of from 1 nucleotide to 100 nucleotides from the single-stranded break in the target DNA.
9 . The system of claim 1 , wherein the endonuclease comprises a class 2 CRISPR/Cas endonuclease.
10 . The system of claim 9 , wherein the class 2 CRISPR/Cas endonuclease comprises a type V or type VI CRISPR/Cas endonuclease.
11 . The system of claim 9 , wherein the class 2 CRISPR/Cas endonuclease comprises a Cas9 polypeptide.
12 . The system of claim 9 , wherein the class 2 CRISPR/Cas endonuclease comprises a Streptococcus pyogenes Cas9 polypeptide.
13 . The system 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 , 10 −4 to 10 −3 , or 10 −3 to 10 −2 mutations per nucleotide per genome replication event.
14 . The system of claim 1 , wherein the fusion polypeptide further comprises a linker connecting the endonuclease and the DNA polymerase.
15 . The system of claim 1 , wherein the fusion polypeptide further comprises a nuclear localization signal.
16 . The system 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.
17 . The system 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.
18 . The system 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.
19 . The system 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.
20 . The system 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.
21 . The system 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.
22 . The system 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.
23 . The system 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.