COMPOSITIONS AND METHODS FOR DELIVERING A NUCLEOBASE EDITING SYSTEM
The invention provides compositions and methods for delivering first and second polynucleotides each encoding a fragment of an A-to-G Base Editor fusion protein comprising one or more deaminases (e.g., adenosine deaminases) and nCas9, wherein the first polynucleotide encodes an N-terminal fragment of nCas9 fused to an intein-N of a split intein pair and the second polynucleotide encodes a C-terminal fragment of nCas9 fused to an intein-C of a split intein pair, and methods for delivering these fragments together with an sgRNA to a cell (e.g., AAV delivery), where the fragments are spliced together by a split intein system, thereby reconstituting a functional base editing system in the cell.
1 . A composition comprising
(a) a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at a position between A292-G364 of Cas9 as numbered in SEQ ID NO: 2, wherein the N-terminal fragment of Cas9 is fused to a split intein-N, and
(b) a second polynucleotide encoding a C-terminal fragment of Cas9, wherein the C-terminal fragment of Cas9 starts at a position between A292-G364 of Cas9 as numbered in SEQ ID NO: 2 and is a contiguous sequence that terminates at the C-terminus of Cas9, and wherein the C-terminal fragment of Cas9 is fused to a split intein-C; or
(a) a first polynucleotide encoding an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at a position between A292-G364 of Cas9 as numbered in SEQ ID NO: 2, wherein the N-terminal fragment of Cas9 is fused to a split intein-N, and
(b) a second polynucleotide encoding fusion protein comprising a C-terminal fragment of Cas9 and a deaminase, wherein the C-terminal fragment of Cas9 starts at a position between A292-G364 of Cas9 as numbered in SEQ ID NO: 2 and is a contiguous sequence that terminates at the C-terminus of Cas9, and wherein the C-terminal fragment of Cas9 is fused to a split intein-C; or
(a) a first polynucleotide encoding a fusion protein comprising a deaminase and an N-terminal fragment of Cas9, wherein the N-terminal fragment of Cas9 starts at the N-terminus of Cas9 and is a contiguous sequence that terminates at a position between A292-G364, F445-K483, or E565-T637 of Cas9 as numbered in SEQ ID NO: 2, wherein the N-terminal fragment of Cas9 is fused to a split intein-N, and
(b) a second polynucleotide encoding a C-terminal fragment of Cas9, wherein the C-terminal fragment of Cas9 starts at a position between A292-G364, F445-K483, or E565-T637 of Cas9 as numbered in SEQ ID NO: 2 and is a contiguous sequence that terminates at the C-terminus of Cas9, wherein the N-terminus residue of the C-terminal fragment of Cas9 is a Cys substituted for an Ala, Ser, or Thr, and wherein the C-terminal fragment of Cas9 is fused to a split intein-C.