Harvard College

The Broad Institute, Inc.

Jonathan Ma Levy

David R Liu

Andrew Vito Anzalone

Xin Gao

Christopher J Podracky

Wei Hsi Yeh

The present disclosure provides systems, compositions, and methods for simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited. In some aspects, the systems comprise a first and second prime editor complex, wherein each of the first and second prime editor complexes comprises (1) a prime editor comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp), and (ii) a polypeptide having an RNA-dependent DNA polymerase activity; and (2) a pegRNA comprising a spacer sequence, gRNA core, a DNA synthesis template, and a primer binding site, wherein the DNA synthesis template encodes a desired DNA sequence or a complement thereof, wherein the desired DNA sequence and the complement thereof form a duplex comprising an edited portion which integrates into the target site to be edited. In some aspects, the systems comprise a first, second, third, and fourth prime editor complex, each comprising a prime editor and a PEgRNA. Also provided herein are methods for simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited. Further provided herein are pharmaceutical compositions, polynucleotides, vectors, cells, and kits.

Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

The present disclosure provides systems, compositions, and methods for simultaneously editing both strands of a double-stranded DNA sequence at a target site to be edited. Further provided herein are pharmaceutical compositions, polynucleotides, vectors, cells, and kits for simultaneously editing both strands of a double-stranded DNA sequence.

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.

Growing community of inventors

Boston, MA, United States of America

Jonathan Ma Levy