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

US12031126B2 · kind B2 · utility

Assignees

• The Broad Institute, Inc. · US
• President and Fellows of Harvard College · US

Inventors

• David R. Liu · Lexington, US
• Andrew Vito Anzalone · Cambridge, US
• Jonathan Ma Levy · Boston, US
• Xin Gao · Chengdu, CN
• Christopher J. Podracky · Cambridge, US

Key dates

• Filing date: Dec 8, 2023
• Grant date: Jul 9, 2024
• Expiry date: Dec 8, 2043

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N2310/3519
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

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, vect…