Target Interaction of Cas9-sgRNA Influences DNA Double Strand Break Repair Pathway Choices in CRISPR/Cas9 Genome Editing

Abstract Background Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double strand breaks (DSBs) have to be exposed in order to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target-binding affinity and modulates its post-cleavage target-residence duration and exposure of Cas9-induced DSBs. This exposure by different mechanisms may initiate variable cellular DNA damage response, thus influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. Results In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical non-homologous end joining (c-NHEJ) is not even engaged at some sites. Weakening target interaction of Cas9-sgRNA promotes bias towards c-NHEJ, which is intrinsically accurate for Cas9-induced DSBs. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ however aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways whereas transcription has little effect on DSB repair pathway choices. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. Conclusions Target interaction of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.