The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations

Mutagenic outcomes of CRISPR/Cas9-generated double-stranded breaks depend on both the sequence flanking the cut and cellular DNA damage repair. The interaction of these features has been largely unexplored, limiting our ability to understand and manipulate the outcomes. Here, we measured how the absence of 18 repair genes changed frequencies of 83,680 unique mutational outcomes generated by Cas9 double-stranded breaks at 2,838 synthetic target sequences in mouse embryonic stem cells. This large scale survey allowed us to classify the outcomes in an unbiased way, generating hypotheses about new modes of double-stranded break repair. Our data indicate a specialised role for Prkdc (DNA-PKcs protein) and Polm (Polμ) in creating 1bp insertions that match the nucleotide on the proximal side of the Cas9 cut with respect to the protospacer-adjacent motif (PAM), a variable involvement of Nbn (NBN) and Polq (Polθ) in the creation of different deletion outcomes, and a unique class of uni-directional deletion outcomes that are dependent on both end-protection gene Xrcc5 (Ku80) and the resection gene Nbn (NBN). We used the knowledge of the reproducible variation across repair milieus to build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of CRISPR/Cas9-generated mutations.