<span class="paragraphSection"><div class="boxTitle">Abstract</div>DNA repair is critical for cellular homeostasis under both normal conditions as well as in response to genotoxic agents such as chemotherapeutics. EXO1 is a 5′–3′ exonuclease with multiple roles in DNA biology. To better understand these roles, we employed CRISPR loss-of-function genome-wide screening to identify genes required for proliferation and cisplatin sensitivity in EXO1-deficient cells. We uncovered differential regulators of cisplatin sensitivity between wildtype (WT) and EXO1-deficient cells. By analyzing the genetic networks that these regulators belong to, we found that DNA repair was the main biological process suppressing cisplatin sensitivity in WT cells, but this was not the case in EXO1-deficient cells, indicating that EXO1 is critical for the repair of cisplatin-induced DNA damage. Moreover, synthetic lethality screens identified a genetic interaction between EXO1 and the histone chaperone CAF-1. Mechanistically, we show that EXO1 and CAF-1 are independently recruited to R-loops and participate in separate, synergistic pathway of R-loop suppression. Even in the absence of DNA damage treatment, concomitant loss of EXO1 and CAF-1 causes R-loop accumulation and increased R-loop-associated DNA damage. Our work sheds light on the critical roles of EXO1 in genomic stability.</span>