Updated project metadata. Over one million cases of gastric cancer are diagnosed each year and metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway creating therapeutic opportunities through synthetic lethal approaches. In particular, several small molecule inhibitors of Ataxia-Telangiectasia Mutated and Rad3-related protein kinase (ATR), a key regulator of the DNA Damage response, are now in clinical development as targeted agents for gastric cancer. Here, we sought to discover genetic determinants of response and resistance to ATRi in gastric cancer cells. We performed a large-scale CRISPR interference screen to discover determinants of resistance to ATRi in gastric cancer. Top hits were validated and further evaluated to define mechanisms of resistance. We identified components of the nonsense-mediated decay pathway and the UPF2 gene, in particular, as mediators of sensitivity to ATRi. We show loss of UPF2 causes resistance to ATRi through the abrogation of ATRi mediated cell cycle arrest and find evidence that UPF2 regulates R-loops. Our results uncover a novel role for NMD factors in R-loop formation and resistance to ATRi in gastric cancer cells, potentially through R-loop formation.