Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Peter J Chen,Jeffrey A Hussmann,Jun Yan,Friederike Knipping,Purnima Ravisankar,Pin-Fang Chen,Cidi Chen,James W Nelson,Gregory A Newby,Mustafa Sahin,Mark J Osborn,Jonathan S Weissman,Britt Adamson,David R Liu

doi:10.1016/j.cell.2021.09.018

Abstract

SummaryWhile 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.

Highlights

The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in the clinical treatment of genetic diseases
Toward the goal of enabling a wide range of sequence changes, we developed prime editing, a versatile gene editing approach that can install all types of targeted DNA base pair substitutions, small insertions, small deletions, and combinations thereof without requiring double-strand DNA breaks (DSBs) or donor DNA templates (Anzalone et al, 2020; Anzalone et al, 2019)
Design of a pooled CRISPR interference (CRISPRi) screen for prime editing outcomes We reasoned that identifying genetic determinants of prime editing sequence outcomes, including the original sequence, the desired edit, and indels, could inform strategies to maximize efficiency and minimize unwanted byproducts

Summary

Introduction

The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in the clinical treatment of genetic diseases. Toward the goal of enabling a wide range of sequence changes, we developed prime editing, a versatile gene editing approach that can install all types of targeted DNA base pair substitutions, small insertions, small deletions, and combinations thereof without requiring double-strand DNA breaks (DSBs) or donor DNA templates (Anzalone et al, 2020; Anzalone et al, 2019). Prime editing minimally requires two components: an engineered reverse transcriptase (RT) fused to Cas nickase (the PE2 protein) and a prime editing guide RNA (pegRNA) that contains both a spacer sequence complementary to target DNA and a 30 extension encoding the desired edit (Anzalone et al, 2019) (Figure 1A).

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Discussion

Conclusion