Cas9-sgRNA target residence alters DNA double strand break repair pathway choices in CRISPR/Cas9 genome editing

Abstract

Given its different target-binding affinity and varying residence time after DNA cleavage, the Cas9-sgRNA complex that remains bound to cleaved DNA may influence DNA double strand break (DSB) repair pathway choices, contributing to highly heterogeneous mutations in genome editing. Here, we found that DSB repair pathway choices vary significantly at different sites for Cas9-induced DSBs. Reduced target-binding affinity of Cas9-sgRNA promotes a bias toward classical NHEJ (c-NHEJ), but inactivation of c-NHEJ aggravates off-target activities. By dislodging Cas9-sgRNA from the cleaved target, local DNA replication suppresses c-NHEJ in favor of other DSB repair pathways by altering DSB configuration and repair timing. It also results in sister chromatid fusion, which may promote chromosomal breakage-fusion-bridge cycles. This suggests that varying Cas9-sgRNA target-binding affinity or residence duration modulate DSB repair pathway choices for Cas9-induced DSBs and widen the mutational spectra in CRISPR/Cas9 genome editing.