MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While tar-geted gene knockout by CRISPR-Cas9 through non-homolo-gous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we opti-mized several key parameters that affect HDR, including ho-mology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchroniza-tion of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43-to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antago-nists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep pri-mary cells.

Automated summary

CRISPR-Cas9-mediated genome editing is useful in sheep for both agricultural and biomedical applications. However, the efficiency of knockin via homology-directed repair (HDR) is lower in sheep fetal fibroblasts (SFFs), hindering precise genome editing. This study optimized key parameters affecting HDR in SFFs and identified small molecules that improved HDR efficiency by activating the p53 pathway. Additionally, genetic knockout of p53 suppressed HDR in SFFs. Overall, this study provides an optimized strategy for dsDNA repair template usage and a simple, efficient method for CRISPR-Cas9-mediated precise genome editing in sheep primary cells.