Efficient CRISPR/HDR-mediated knock-in system and method of use

US11530424B1 · kind B1 · utility

Assignees

• University of South Florida · US
• United States Department of Veteran Affairs · US

Inventors

• Jia-Wang Wang · Tampa, US
• Richard F. Lockey · Tampa, US

Key dates

• Filing date: Sep 30, 2019
• Grant date: Dec 20, 2022
• Expiry date: Mar 23, 2041

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N2310/20
• WIPO field: Pharmaceuticals
• WIPO sector: Chemistry

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, can efficiently generate gene knockouts of variably sizes. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. HDR-mediated gene knock-in experiments are inefficient, with no reports of successful gene knock-in with DNA fragments larger than 4 kb. Targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors was performed and indicate that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected.