Improving Gene Knock-In Efficiencies in Sheep Primary Cells Using a CRISPR/Cas9-Gal4 System.

ABSTRACT: Currently, a major challenge exists in CRISPR-mediated genome editing research in sheep: the low efficiency of exogenous large DNA fragment targeted integration without drug selection or fluorescence enrichment. This restriction significantly impedes the use of precise genome editing in sheep for agricultural, biological, and biomedical purposes. In this study, we employed the strategy of increasing the local concentration of the homologous repair template at the site of the DNA double-strand break (DSB). We achieved this by fusing the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein using a 32-amino acid (aa) flexible linker. Additionally, we incorporated a 17 bp UAS at the 3' end of the donor template, which can be specifically recognized

SUBMITTER: Li Y 

PROVIDER: S-EPMC12651790 | biostudies-literature | 2025 Oct

REPOSITORIES: biostudies-literature