ABSTRACT Recent studies have revealed a role for RNA in the repair of DNA double-strand breaks. Here, we show that the asymmetric DNA overhangs generated by the small TevSaCas9 dual nuclease informs a simple and robust editing strategy in human cells whereby Polθ and Rad52 are recruited to repair the double-strand break. The 2-nt, 3’ DNA overhang generated by the I-TevI nuclease domain of TevSaCas9 hybridizes with the 3’ end of a co-localized repair template guide RNA to specifically license repair. Substitutions that destabilize the repair duplex reduce editing efficiency. Targeted RNA-templated repair (rep-editing) harnesses cellular RNA-based DNA repair pathways to introduce precise nucleotide edits, deletions and insertions in human cells with high efficiency and fidelity independent of co-delivered repair functions. The small size of TevSaCas9 and RNA repair template offers delivery advantages over size-constrained or multi-component editing systems.

Harnessing RNA-based DNA repair pathways for targeted gene editing