Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.
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.
|Year of Publication
2021 10 28
|PubMed Central ID
R01 HL156647 / HL / NHLBI NIH HHS / United States
R35 GM138167 / GM / NIGMS NIH HHS / United States
P30 CA072720 / CA / NCI NIH HHS / United States
RM1 HG009490 / HG / NHGRI NIH HHS / United States
HHMI / Howard Hughes Medical Institute / United States
R35 GM118062 / GM / NIGMS NIH HHS / United States
U01 AI142756 / AI / NIAID NIH HHS / United States
U54 HD090255 / HD / NICHD NIH HHS / United States
R01 EB031172 / EB / NIBIB NIH HHS / United States
P50 HD105351 / HD / NICHD NIH HHS / United States
P01 CA065493 / CA / NCI NIH HHS / United States