RNA transcripts serve as a template for double-strand break repair in human cells.

Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, growing evidence suggests that RNA:DNA hybrids and nearby transcripts can influence repair outcomes. However, whether transcript RNA can directly serve as a template for DSB repair in human cells remains unclear. In this study, we develop fluorescence and sequencing-based assays to show that RNA-containing oligonucleotides and messenger RNA can serve as templates during DSB repair. We conduct a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR). Of the candidate polymerases, we identify DNA polymerase zeta (Polζ) as a potential reverse transcriptase that facilitates RT-DSBR. Furthermore, analysis of cancer genome sequencing data reveals whole intron deletions - a distinct genomic signature of RT-DSBR that occurs when spliced mRNA guides repair. Altogether, our findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.