Target residence 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 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, via different mechanisms, may initiate variable DNA damage responses, 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 nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ 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 c-NHEJ engagement. 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 residence 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.

Uncovering the Dynamics of Precise Repair at CRISPR/Cas9-induced Double-Strand Breaks

“…Studies of DSBs induced by CRISPR/Cas9 in endogenous chromatin have primarily focused on mutagenic repair, revealing many aspects affecting editing outcome and repair-pathway choice 41,42 . In addition, kinetic studies have provided insight into the dynamics of the process of induction and error-prone repair [43][44][45][46] .…”

“…Studies of DSBs induced by CRISPR/Cas9 in endogenous chromatin have primarily focused on mutagenic repair, revealing many aspects affecting editing outcome and repair-pathway choice 41,42 . In addition, kinetic studies have provided insight into the dynamics of the process of induction and error-prone repair 43–46 . However, as a result of the inability to directly observe scar-less re-ligation, measuring the degree of precise repair, particularly by the end-joining pathways, remains challenging.…”

Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining

“…Fusion of HDR facilitators to the widely used Cas nuclease Streptococcus pyogenes Cas9 ( Sp Cas9) and arresting the cell cycle at the S/G2 phase by chemicals have improved HDR-mediated CRISPR genome editing ( 3 , 9 , 12–18 ). Chemical inhibition or genetic inactivation of c-NHEJ, the most dominant competing pathway against HDR, could channel DSBs that are supposedly repaired by c-NHEJ to HDR, thereby increasing the efficiency of HDR in repair of Cas nuclease-induced DSBs ( 3 , 8 , 9 ). Indeed, inhibition of c-NHEJ is often used to promote HDR-mediated genome editing ( 3 , 9 , 19–22 ).…”

“…Indeed, inhibition of c-NHEJ is often used to promote HDR-mediated genome editing ( 3 , 9 , 19–22 ). However, due to global inhibition of c-NHEJ, our recent study demonstrated that this approach unavoidably exacerbates off-target effect ( 8 ). We reason this off-target problem could be solved by local inhibition of c-NHEJ; but such a strategy has yet to be developed.…”

“…Cas9-induced DSBs are exposed after target dissociation of Cas9–sgRNA. As exposure of Cas9-induced DSBs is prerequisite for engaging DSB repair, target residence of Cas9–sgRNA adds a layer of control on pathway choices in repair of Cas9-induced DSBs, contributing to the heterogeneity of CRISPR/Cas9 genome editing ( 8 , 24 ). The similarly persistent binding of nuclease dead Sp Cas9 (d Sp Cas9) to its target is capable to slow or block DNA replication and transcription in cells ( 27–29 ).…”