Dissecting mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform wholegenome sequencing of 173 daughter subclones. Δ, Δ, Δ, Δ, Δ, Δ, Δ, Δ, and Δ produce marked mutational signatures indicative of being critical mitigators of endogenous DNA changes. Detailed analyses reveal that 8-oxo-dG removal by different repair proteins is sequence-context-specific while uracil clearance is sequencecontext-independent. Signatures of mismatch repair (MMR) deficiency show components of C>A transversions due to oxidative damage, T>C and C>T transitions due to differential misincorporation by replicative polymerases, and T>A transversions for which we propose a ‘reverse template slippage’ model. Δ, Δ, and Δ signatures are similar to each other but distinct from Δ. We validate these gene-specificities in cells from patients with Constitutive Mismatch Repair Deficiency Syndrome. Based on these experimental insights, we develop a classifier, MMRDetect, for improved clinical detection of MMR-deficient tumors.