Nucleobase editors having reduced non-target deamination and assays for characterizing nucleobase editors

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]-[TadA adenosine 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, as numbered in SEQ ID NO:
• 1,
• wherein the TadA adenosine deaminase is inserted at the deletion position, and wherein the TadA adenosine deaminase of the fusion protein deaminates a target nucleobase in a target DNA molecule and effects reduced deamination at non-target sites compared with an end-terminus fusion protein comprising the TadA adenosine deaminase fused to an N-terminus or C-terminus of a Cas9 polypeptide.

A polynucleotide encoding a fusion protein, wherein the fusion protein comprises:

• A) a TadA adenosine 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 TadA adenosine deaminase is inserted between amino acid positions 768-769, 1022-1023, 1026-1027, 1040-1041, 1068-1069, or 1247-1248, 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]-[TadA adenosine deaminase]-[C-terminal fragment of a Cas91-COOH, wherein each instance of “1-” is an optional linker,
• wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, as numbered in SEQ ID NO:
• 1,
• wherein the TadA adenosine deaminase is inserted at the deletion position;
• and
• wherein the TadA adenosine deaminase of the fusion protein deaminates a target nucleobase in a target DNA molecule and effects reduced deamination at non-target sites compared with an end-terminus fusion protein comprising the TadA adenosine deaminase fused to an N-terminus or C-terminus of a Cas9 polypeptide.

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 TadA adenosine deaminase inserted within 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 Cas91-COOH, wherein each instance of “1-” is an optional linker,
• wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, as numbered in SEQ ID NO:
• 1,
• wherein the TadA adenosine deaminase is inserted at the deletion position, and wherein the TadA adenosine deaminase of the fusion protein effects reduced deamination at non-target sites compared with an end-terminus fusion protein comprising the TadA adenosine deaminase fused to an N-terminus or C-terminus of a Cas9 polypeptide.

A method for treating a genetic condition in a subject, the method comprising:

• administering to the subject a fusion protein comprising a TadA adenosine deaminase inserted within a Cas9 polypeptide, wherein the fusion protein comprises the structure:
• NH2-[N-terminal fragment of a Cas91-[TadA adenosine deaminase]-[C-terminal fragment of a Cas9]-COOH, wherein each instance of “1-” is an optional linker,
• wherein the Cas9 polypeptide comprises a deletion of amino acid positions 794-905, as numbered in SEQ ID NO:
• 1,
• wherein the TadA adenosine deaminase is inserted at the deletion position, 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, and wherein the TadA adenosine deaminase effects reduced deamination at non-target sites compared with an end-terminus fusion protein comprising the TadA adenosine deaminase fused to an N-terminus or C-terminus of a Cas9 polypeptide, wherein deamination of the target nucleobase ameliorates symptoms of the genetic condition, thereby treating the genetic condition.