AAV delivery of nucleobase editors
Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated virus (rAAV), to form a complete and functional Cas9 protein or nucleobase editor. The Cas9 protein or the nucleobase editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Recombinant AAV vectors and particles for the delivery of the split Cas9 protein or nucleobase editor, and methods of using such AAV vectors and particles are also provided.
1 . A composition comprising:
(i) a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and
(ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein;
wherein the N-terminal portion and the C-terminal portion may be joined to form a complete Cas9 protein; and
wherein the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a nucleobase modifying enzyme fused to the N-terminus of the N-terminal portion of the Cas9 protein.
2 . The composition of claim 1 , wherein the N-terminal portion of the Cas9 protein comprises the portion of any one of SEQ ID NOs: 1-275 and 394-397 that corresponds to amino acids 1-573 or 1-637 of SEQ ID NO: 1.
3 . The composition of claim 1 , wherein the C-terminal portion of the Cas9 protein comprises the portion of any one of SEQ ID NOs: 1-275 and 394-397 that corresponds to amino acids 574-1368 or 638-1368 of SEQ ID NO: 1.
4 . The composition of claim 1 , wherein the intein-C comprises the amino acid sequence as set forth in SEQ ID NO: 357.
5 . The composition of claim 1 , wherein the first nucleotide sequence or the second nucleotide sequence further comprises a nucleotide sequence encoding a guide RNA (gRNA) operably linked to a promoter.
6 . The composition of claim 1 , wherein the first nucleotide sequence and/or the second nucleotide sequence further comprises a transcriptional terminator.
7 . The composition of claim 6 , wherein the transcriptional terminator is the transcriptional terminator from a bGH gene, hGH gene, or SV40 gene.
8 . The composition of claim 6 , wherein the first nucleotide sequence and/or the second nucleotide sequence further comprises a woodchuck hepatitis posttranscriptional regulatory element (WPRE) inserted 5′ of the transcriptional terminator.
9 . The composition of claim 1 , wherein the Cas9 protein is a catalytically inactive Cas9 (dCas9).
10 . The composition of claim 1 , wherein the Cas9 protein is a Cas9 nickase (nCas9).
11 . The composition of claim 1 , wherein the nucleobase modifying enzyme is a deaminase, DNA repair enzyme, DNA damage repair enzyme, alkylation enzyme, depurination enzyme, oxidation enzyme, pyrimidine dimer-forming enzyme, photolyase, glycosylase, acetylase, methyltransferase, or demethylase.
12 . The composition of claim 1 , wherein the first nucleotide sequence and the second nucleotide sequence are on different vectors.
13 . The composition of claim 12 , wherein each of the different vectors is a genome of a recombinant adeno-associated virus (rAAV).
14 . The composition of claim 13 , wherein each of the different vectors is packaged in an rAAV particle.
15 . A method comprising contacting a cell with the composition of claim 1 , wherein the contacting results in the delivery of the first nucleotide sequence and the second nucleotide sequence into the cell.
16 . The composition of claim 1 , wherein the Cas9 protein comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 2 or 3.
17 . The composition of claim 1 , wherein the Cas9 protein comprises the amino acid sequence of SEQ ID NO: 2 or 3.
18 . The composition of claim 1 , wherein the first nucleotide sequence and/or second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal.
19 . The composition of claim 18 , wherein the at least one bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of:
(SEQ ID NO: 344)
KRPAATKKAGQAKKKK,
(SEQ ID NO: 345)
KKTELQTTNAENKTKKL,
(SEQ ID NO: 346)
KRGINDRNFWRGENGRKTR,
and
(SEQ ID NO: 347)
RKSGKIAAIVVKRPRK.
20 . The composition of claim 12 , wherein the intein-N comprises the amino acid sequence as set forth in SEQ ID NO: 355.
21 . The composition of claim 7 , wherein the transcriptional terminator is from a bGH gene.
22 . The composition of claim 1 , wherein the nucleobase modifying enzyme fused to the N-terminal portion of the Cas9 protein and the C-terminal portion of the Cas9 protein may be joined to form a complete Cas9-fusion protein editor.
23 . A composition comprising:
(i) a first recombinant adeno associated virus (rAAV) particle comprising a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and
(ii) a second recombinant adeno associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of a C-terminal portion of the Cas9 protein;
wherein the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a nucleobase modifying enzyme fused to the N-terminus of the N-terminal portion of the Cas9 protein; and
wherein the N-terminal portion and the C-terminal portion may be joined to form a complete Cas9 protein.
24 . A method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 23 .
25 . The composition of claim 23 , wherein the intein-C comprises the amino acid sequence of SEQ ID NO: 357.
26 . The composition of claim 23 , wherein the intein-N comprises the amino acid sequence as set forth in SEQ ID NO: 355.
27 . An isolated cell comprising the composition of claim 1 or 23 .
28 . The isolated cell of claim 27 further comprising the N-terminal portion of the Cas9 protein encoded by the first nucleotide sequence of the composition and the C-terminal portion of the Cas9 protein encoded by the second nucleotide sequence of the composition joined together to form the complete Cas9 protein, wherein the N-terminus of the N-terminal portion of the Cas9 protein is fused to the nucleobase modifying enzyme.
29 . The isolated cell of claim 27 , wherein the isolated cell is a human cell.