A mutational scar-based genome-wide map of DNA double-strand break repair

Genome alterations arise from inaccurate DNA repair and accumulate as distinct mutational signatures. Here, we systematically interrogate the contribution of every protein-coding gene to double-strand break (DSB) repair by generating high-resolution outcome profiles following gene knockouts. Using a CRISPR/Cas9-based, massively parallel bulk screening approach, we establish a comprehensive catalogue of MUtational Scars of Induced DNA Cleavage (MUSIC) that maps the full landscape of DSB repair factors. Our analysis identifies and validates gene clusters – including nearly all known components and several previously unrecognised factors – associated with non-homologous end-joining, the 53BP1 pathway, homology-directed repair, and polymerase theta (POLQ)-mediated end-joining. By focusing on pathway-specific repair outcomes, we uncover an unexpected role for the WRN helicase in suppressing inverted templated insertions, a poorly understood POLQ-associated mutational signature. Finally, dissection of MUSIC features reveals unanticipated functional distinctions among genes within the same DSB pathway, providing mechanistic insight and enabling further investigation into chromosomal break repair. Mutational scars act as biomarkers for inaccurate DNA repair. Here, the authors systematically interrogate the contribution of every protein-coding gene to DNA double-strand break repair by generating outcome profiles following gene knockouts to extract mechanistic insight from profile alterations.