Cas12a-based combinatorial knockout screens define the genetic architecture of the DNA damage response

To elucidate the genetic architecture of the DNA damage response (DDR) network, we conduct unbiased genetic interaction screens using Cas12a. We disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations, enabling us to assess the phenotypic effects induced by the loss of DDR gene pairs under unperturbed cell growth conditions. We identify high-confidence positive and negative gene-gene interactions, along with connections between previously unlinked DDR pathways. Among the identified genetic interactions, we uncover synthetic lethal interactions between genes encoding the translesion polymerase REV1-Pol ζ complex and the MCM8-MCM9-HROB DNA helicase complex, between Fanconi anemia genes and various mitotic DNA repair genes, as well as between genes encoding the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting opportunities for targeted treatments in tumors carrying mutations in these genes. Collectively, our genetic map of the DDR network identifies synthetic lethal and buffering interactions with therapeutic potential.