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

Published: 22 February 2021| Version 2 | DOI: 10.17632/ymn3ykkmyx.2
Serena Nik-Zainal


Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts (∆) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform whole-genome sequencing of 173 daughter subclones. ∆OGG1, ∆UNG, ∆EXO1, ∆RNF168, ∆MLH1, ∆MSH2, ∆MSH6, ∆PMS1, and ∆PMS2 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 sequence-context-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. ∆MLH1, ∆MSH6, and ∆MSH2 signatures are similar to each other but distinct from ∆PMS2. 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, to detect MMR-deficient tumors. Application of MMRDetect to 7359 WGS cancers shows improved clinical detection of patients with MMR deficient whom could have responsiveness to immunotherapy agents.