Developed PE4 and PE5 variants with MLH1dn fusion to evade mismatch repair. Achieved 2-6 fold efficiency improvement. Established cellular pathway manipulation for editing enhancement.
Enhanced prime editing systems by manipulating cellular determinants of editing outcomes
Prime editing efficiency varied unpredictably across cell types for unclear reasons. CRISPRi screens found mismatch repair suppresses editing, so the authors added dominant-negative MLH1 to build PE4/PE5. These raised efficiency 7.7-fold over PE2 and 2-fold over PE3.
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Key findings
Abstract
While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.