Poly(ADP-ribose) polymerase inhibitors (PARPi) are extensively explored as tumor-targeted therapies for homologous recombination (HR)-deficient cancers because of the observed synthetic lethality between PARP inhibition and BRCA1/2 mutations. The cytotoxicity of PARPi is mainly caused by the trapping of PARP1 on DNA, thus interfering with DNA duplication. To catalog cellular PARPi resistance genes, Zimmermann et al. utilized a CRISPR dropout screen approach and identified components of the RNase H2 enzyme complex as top hits. The subsequent mechanistic study revealed that the protective effect of RNase H2 was mediated by its ability to remove genome-embedded ribonucleotides without affecting HR efficiency. In addition, depleting cellular TOP1, an enzyme that can also cleave DNA-embedded ribonucleotides, reduced the DNA damage induced by the absence of RNase H2 and restored the resistance to PARPi in RNase-H2-null cells. These results suggested that TOP1-mediated ribonucleotide processing leads to DNA lesions that recruit PARP1, sensitizing RNase-H2-deficient cells to PARP trapping. This study reveals how DNA-embedded ribonucleotides cause DNA damage and PARPi sensitivity and lays a mechanistic foundation for the potential clinical use of PARPi in patients with RNase H2 deficiency.