Role of small non-coding RNAs in DSB repair in Arabidopsis thaliana

DNA double-strand breaks (DSBs) are toxic lesions that must be repaired to avoid genetic loss and genome rearrangements. Various conserved pathways are involved in DSB repair, producing different outcomes. Accumulating evidence has identified RNA, particularly small RNAs as a new player in DSB repair. DSB-induced small RNAs (diRNAs) have been detected at DSB-flanking sequences using reporter systems, but their endogenous occurrence and function in DNA repair remain debated. Here, we investigated diRNA biogenesis in Arabidopsis thaliana using CRISPR/Cas12a to induce DSBs at three transcribed loci and one untranscribed locus. We found that 21-nt diRNAs are generated at transcribed loci, in a Dicer-like 4 (DCL4)-dependent manner, but not at the untranscribed locus, indicating that active transcription is required for diRNA production. Long-read DNA sequencing analysis showed that diRNAs do not detectably influence DSB repair via either end-joining or microhomology-mediated end-joining. Furthermore, endogenous diRNAs have a modest effect on homologous recombination-associated repair, as impairment of the diRNA pathway in ago2 mutants caused only a slight reduction in RAD51 focus formation following Cas12a-induced DSBs. Together, these findings indicate that diRNAs are not essential for DSB repair but may function in post-transcriptional silencing of damaged genes, potentially preventing aberrant transcription until repair is completed.