CRISPR screening identifies SMARCAL1 and MRN as modulators of WRN dependency in MSI-H colorectal cancer

Microsatellite instability-high (MSI-H) colorectal cancers (CRCs) rely on the Werner syndrome helicase (WRN) to resolve cruciform DNA structures that arise from expanded TA-dinucleotide repeats. Pharmacologic inhibition of WRN with the selective small molecule HRO761 recapitulates WRN loss, causing replication stress and double-strand breaks (DSBs). However, mechanisms modulating WRN dependency remain incompletely understood. To systematically identify genetic determinants of WRN inhibitor sensitivity, we performed genome-wide CRISPR/Cas9 knockout screens in MSI-H colorectal cancer cell lines treated with or without HRO761. Top candidates were validated using individual gene knockouts, RAD50 degron (dTAG) knock-in systems, and biochemical and imaging assays for DNA damage and replication stress. SMARCAL1 emerged as the strongest resistance hit, and its depletion conferred a 3-5-fold reduction in HRO761 sensitivity and attenuated gammaH2AX induction. SMARCAL1 likely antagonizes WRN helicase activity and promotes cruciform DNA stabilization via its annealing helicase function. In contrast, loss of structure-specific nucleases MUS81 or ERCC1-XPF did not affect HRO761 response. Instead, acute degradation of RAD50 disrupted the MRE11/RAD50/NBS1 (MRN) complex and conferred ~10-fold resistance to WRN inhibition, accompanied by reduced DSB signaling. ATM, recruited by the MRN complex, also mediated WRN inhibitor sensitivity, as ATM knockout similarly induced resistance. Our results identify SMARCAL1 and the MRN-ATM axis as critical regulators of WRN dependency in MSI-H CRC. These findings redefine the mechanism of WRN synthetic lethality and provide a framework for predicting and overcoming resistance to emerging WRN-targeted therapies.