Prime Editing Efficiency and Fidelity are Enhanced in the Absence of Mismatch Repair

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.