A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR–Cas9 knockouts (∆) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage and performed whole-genome sequencing of 173 subclones. ∆ OGG1 , ∆ UNG , ∆ EXO1 , ∆ RNF168 , ∆ MLH1 , ∆ MSH2 , ∆ MSH6 , ∆ PMS1 and ∆ PMS2 produced marked mutational signatures indicative of them being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-2′-deoxyguanosine elimination is sequence context specific while uracil clearance is sequence context independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C > A transversions) and differential misincorporation by replicative polymerases (T > C and C > T transitions), and we propose a reverse template slippage model for T > A transversions. ∆ MLH1 , ∆ MSH6 and ∆ MSH2 signatures were similar to each other but distinct from ∆ PMS2 . Finally, we developed a classifier, MMRDetect, where application to 7,695 whole-genome-sequenced cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.