Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. Here, to determine which cellular factors affect PE efficiency, we carry out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2–17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.

Prime editing is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions, into any given genomic locus. But prime editing, at even the same locus, can exhibit wildly different efficiencies in various cell backgrounds. It is unclear what determines these variations in efficiencies in a given cellular context. Through a focused genetic screen targeting DNA repair factors, we show that the efficiency of prime editing is attenuated by the mismatch repair pathway. The accumulation of the mismatch repair protein MLH1 at sites of prime editing, indicates that mismatch repair acts at these regions to directly counteract the insertion of the edit. Consequently, ablation of mismatch repair yields an up to 17-fold increase in prime editing efficiency across different human cell lines, several types of edits and multiple genomic loci. Our results shed new light on the cellular requirements for prime editing and identify that ablation of mismatch repair increases editing efficiency and fidelity.