Mutational signatures are patterns of mutations that arise during tumorigenesis. We present an enhanced, practical framework for mutational signature analyses. Applying these methods on 3,107 whole genome sequenced (WGS) primary cancers of 21 organs reveals known signatures and nine previously undescribed rearrangement signatures. We highlight inter-organ variability of

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) 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. Δ OGG1, Δ UNG, Δ EXO1, Δ RNF168, Δ MLH1, Δ MSH2, Δ MSH6, Δ PMS1, and Δ PMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG 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), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a ‘reverse template slippage’ model for T>A transversions. Δ MLH1, Δ MSH6, and Δ MSH2 signatures were similar to each other but distinct from Δ PMS2 . Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.