Signatures in CRISPR Mutational Spectra Reveal Role and Interplay of Genes in DNA Repair

MotivationUnderstanding double-strand break (DSB) repair and its disruption is key to decipher genomic instability driving diseases such as cancer and reveal therapeutic avenues. Numerous genes have been linked to DSB repair for the first time in recent genome-wide perturbation studies assessing effects on mutational outcomes. However, the functional roles of most such genes remain poorly understood. Evidence from other studies shows that related genes similarly modulate the frequency of specific mutational outcomes following DSB repair, but analysis has largely ignored the multiplicity of gene functions and cross-talk between pathways. Here, we infer functional roles for candidate genes based on knockout mutational spectra by identifying mutational signatures shared with known genes and then mapping them to DSB repair functions. Signatures are identified using non-negative matrix factorization (NMF) to reflect functional multiplicity and cross-talk. ResultsWe generated mutational spectra for mouse embryonic stem (mES) cells at three target sites across CRISPR knockouts of 307 known and 459 candidate DSB repair genes. Analysis using NMF revealed four mutational signatures associated with homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the initiation and ligation components of non-homologous end-joining (NHEJ). Signatures suggested that candidate genes Dbr1 and Hnrnpk could influence MMEJ and Fanconi anaemia (FA), and that loss of core NHEJ components (e.g. MRN complex or Ku proteins) could shift repair preference towards Ku-independent NHEJ. These findings demonstrate the utility of NMF for characterizing the contribution of genes to repair pathways and provide a foundation to discover new gene functions in DSB repair. Availabilitygithub.com/joanagoncalveslab/MuSpectraNMF.