AAV delivery of nucleobase editors
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, Jonathan Ma Levy, Wei Hsi Yeh
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Abstract
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.
Claims29
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.
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.
Dependencies are inferred from the claim text.