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, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT- DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Pol?) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT- DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT- DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences. Overall design: Amplicon sequencing of adjacent exons to identify if the intron they flank is removed from the genomic sequence. This whole intron deletion serves to measure repair templated by the endogenous spliced mRNA after a DSB within the intron. 293T TP53BP1-/- cells were analyzed at: introns of highly transcribed loci CALR intron 2 and GNAS intron 11, nontranscribed loci IL3 intron 4, adjacent introns, and in a REV3L knockdown derivative.