bioRxiv : the preprint server for biologyJun 22, 2026

Mapping genes involved in DNA damage response using Cas12a gene knockout screens

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 24, 2026

Abstract

Over 750 high-confidence gene-gene interactions were identified from the analysis of more than 27,000 combinations of DNA damage response genes.

The study assessed the effects of disrupting 233 DNA damage response genes, revealing significant interactions among them.
Multiple previously unlinked pathways within the DNA damage response network were connected through these interactions.
Synthetic lethal interactions were found between the REV1-Pol ζ complex and the MCM8-MCM9-HROB helicase complex.
Interactions involving Fanconi Anemia proteins and mitotic DNA repair factors were also identified, indicating potential therapeutic targets.
Robust suppressor interactions were observed between the APOLLO nuclease and core non-homologous end joining genes.
These findings may offer insights into targeted therapies for tumors with specific genetic mutations.

Simplified

The DNA damage response (DDR) is a complex network of cellular pathways that ensures the faithful maintenance of our genomes upon a wide array of genomic insults. To elucidate the functional architecture of this network, we conducted unbiased genetic interaction screens using the Cas12a genome editor to disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations. This approach enabled us to assess the phenotypic effects induced by the disruption of >27,000 DDR gene pair combinations under unperturbed cell growth conditions. From this analysis, we identified over 750 high-confidence positive (buffering) or negative (synthetic lethal/sick) gene-gene interactions, along with multiple connections between previously unlinked DDR pathways and modules, allowing us to define novel aspects of the cellular response to spontaneous, DNA replication-associated DNA damage. Among the identified genetic interactions, we uncovered profound synthetic lethal interactions between genes encoding 1) the translesion polymerase REV1-Pol ζ complex and the MCM8-MCM9-HROB DNA helicase complex; 2) Fanconi Anemia (FA) proteins and the mitotic DNA repair factors GEN1, CIP2A, and RHINO; and 3) the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting novel opportunities for targeted therapies in tumors carrying mutations in these genes. Additionally, we identified robust suppressor interactions between the DCLRE1B gene encoding the nuclease APOLLO and the core non-homologous end joining (NHEJ) genes XRCC4 , LIG4 , and NHEJ1 , suggesting that NHEJ impairs the fitness of APOLLO-deficient cells. This work provides a functional map of the DDR network and demonstrates the power of Cas12a-based screens for identifying synthetic lethal and buffering interactions with therapeutic potential.