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.