Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines.

ABSTRACT: The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, w

SUBMITTER: Liu Z 

PROVIDER: S-EPMC11508559 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature