NUCLEOBASE EDITORS HAVING REDUCED NON-TARGET DEAMINATION AND ASSAYS FOR CHARACTERIZING NUCLEOBASE EDITORS
The invention features base editors having reduced non-target deamination, methods of using the base editors, and assays for characterizing base editors as having decreased non-target deamination, e.g. compared to programmed, on-target deamination.
1 . (canceled)
2 . A fusion protein comprising a deaminase inserted within a flexible loop of a Cas9 polypeptide, wherein the fusion protein comprises the structure:
NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH,
wherein each instance of “]-[” is an optional linker, and wherein the flexible loop comprises a region selected from the group consisting of amino acid residues 768-793, 1002-1040, 1052-1077, and 1232-1248, as numbered in SEQ ID NO: 1.
3 . The fusion protein of claim 2 , wherein the deaminase comprises a TadA adenosine deaminase and is inserted between amino acid positions 768-769, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1054-1055, 1068-1069, or 1247-1248 as numbered in SEQ ID NO: 1.
4 . The fusion protein of claim 2 , wherein the deaminase comprises an APOBEC cytidine deaminase and is inserted between amino acid positions 1016-1017, 1023-1024, 1029-1030, or 1069-1070 as numbered in SEQ ID NO: 1.
5 . A fusion protein comprising a deaminase inserted within a Cas9 polypeptide, wherein the fusion protein comprises the structure:
NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “]-[” is an optional linker,
wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, 792-906, or 792-872 as numbered in SEQ ID NO: 1,
and wherein the deaminase is inserted at the deletion position.
6 . The fusion protein of claim 2 further comprising a nuclear localization signal.
7 . The fusion protein of claim 2 , wherein the fusion protein is in complex with a guide nucleic acid to effect deamination of a target nucleobase.
8 . A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:
A) a deaminase inserted within a flexible loop of a Cas9 polypeptide, wherein the fusion protein comprises the structure:
NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH, and
wherein each instance of “]-[” is an optional linker, wherein the flexible loop comprises a region selected from the group consisting of amino acid residues 768-793, 1002-1040, 1052-1077, and 1232-1248, as numbered in SEQ ID NO: 1; or
B) a deaminase inserted within a Cas9 polypeptide, wherein the fusion protein comprises the structure:
NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “]-[” is an optional linker,
wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, 792-906, or 792-872 as numbered in SEQ ID NO: 1, and
wherein the deaminase is inserted at the deletion position.
9 . An expression vector comprising the polynucleotide of claim 8 .
10 . A cell comprising the fusion protein of claim 2 .
11 . A kit comprising the fusion protein of claim 2 .
12 . A method for base editing comprising contacting a polynucleotide sequence with the fusion protein of claim 2 , wherein the deaminase of the fusion protein deaminates a nucleobase in the polynucleotide, thereby editing the polynucleotide sequence.
13 . A method for editing a target nucleobase in a target polynucleotide sequence, the method comprising: contacting the target polynucleotide sequence with a fusion protein comprising a deaminase inserted within a Cas9 polypeptide, wherein the fusion protein comprises the structure:
NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “]-[” is an optional linker,
wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, 792-906, or 792-872 as numbered in SEQ ID NO: 1, and
wherein the deaminase is inserted at the deletion position.
14 . A method for editing a target nucleobase in a target polynucleotide sequence, the method comprising: contacting the target polynucleotide sequence with a fusion protein comprising a deaminase inserted within a flexible loop of a Cas9 polypeptide, wherein the fusion protein comprises the structure NH2-[N-terminal fragment of a Cas9]-[TadA adenosine deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “]-[” is an optional linker, wherein the deaminase of the fusion protein deaminates the target nucleobase in the target polynucleotide sequence, wherein the flexible loop comprises a region selected from the group consisting of amino acid residues 768-793, 1002-1040, 1052-1077, and 1232-1248, as numbered in SEQ ID NO: 1.
15 . The method of claim 14 , wherein the TadA adenosine deaminase is inserted between amino acid positions 768-769, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1054-1055, 1068-1069, or 1247-1248 as numbered in SEQ ID NO: 1.
16 . The method of claim 12 , wherein the contacting is performed in a cell.
17 . A method for treating a genetic condition in a subject, the method comprising: administering to the subject a fusion protein comprising a deaminase inserted within a Cas9 polypeptide, wherein the fusion protein comprises the structure:
NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “]-[” is an optional linker,
wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, 792-906, or 792-872 as numbered in SEQ ID NO: 1,
wherein the deaminase is inserted at the deletion position, or a polynucleotide encoding the fusion protein, and a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid directs the fusion protein to deaminate a target nucleobase in a target DNA molecule of the subject, wherein the genetic condition is caused by the target nucleobase, which comprises a single nucleotide polymorphism or point mutation at a single locus in the target DNA molecule of the subject.
18 . A method for treating a genetic condition in a subject, the method comprising: administering to the subject a fusion protein comprising a deaminase inserted within a flexible loop of a Cas9 polypeptide, wherein the fusion protein comprises the structure NH2-[N-terminal fragment of a Cas9]-[deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “]-[” is an optional linker, wherein the deaminase of the fusion protein deaminates a target nucleobase in a target DNA molecule of the subject, wherein the C-terminus of the N terminal fragment and the N-terminus of the C terminal fragment comprises a part of the flexible loop of the Cas9 polypeptide, wherein the flexible loop comprises a region selected from the group consisting of amino acid residues 768-793, 1002-1040, 1052-1077, and 1232-1248, as numbered in SEQ ID NO: 1, and a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid directs the fusion protein to deaminate a target nucleobase in a target DNA molecule of the subject, wherein the genetic condition is caused by the target nucleobase, which comprises a single nucleotide polymorphism or point mutation at a single locus in the target DNA molecule of the subject, wherein deamination of the target nucleobase ameliorates symptoms of the genetic condition, thereby treating the genetic condition.
19 . The method of claim 17 , wherein the deamination of the target nucleobase replaces the target nucleobase with a wild type nucleobase.
20 . The method of claim 17 , wherein the deamination of the target nucleobase replaces the target nucleobase with a non-wild type nucleobase.