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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.

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.

Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, unbalanced translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.

Primary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer. Here, using this approach targeting 34 tumor suppressor genes (TSGs), we efficiently induced autochthonous liver tumors in mice within 30-60 days. Analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of intratumor genetic heterogeneity (IGH) in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of extrachromosomal circular DNA (eccDNA) harboring targeted TSG mutations in these tumor cells. These findings suggest an important contribution of eccDNA carrying TSG mutations to tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.