The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy. Display omitted •Resource of 31 genome-scale CRISPR screens against DNA-damaging agents•Cytotoxicity of G-quadruplex ligand pyridostatin involves TOP2 trapping•The bone-marrow failure syndrome gene ERCC6L2 codes for an NHEJ factor•The ELOF1 and STK19 proteins are candidate TC-NER factors A set of CRISPR screens in cells treated with different genotoxic agents illuminates the cellular response to DNA damage, identifying new factors in several repair pathways and pinpointing a novel drug mechanism-of-action.