哈佛大学和麻省理工学院布罗德研究所David R. Liu研究组通过操纵编辑结果的细胞决定因素来增强引导编辑系统。2021年10月14日出版的《细胞》杂志发表了这一最新研究成果。

使用汇集CRISPRi 筛选，他们发现 DNA 错配修复 (MMR) 阻碍了引导编辑并促进了不需要的插入缺失副产物。他们开发了 PE4 和 PE5 引导编辑系统，其中与 PE2 和 PE3 系统相比，工程化 MMR 抑制蛋白的瞬时表达将替换、小插入和小缺失引导编辑的效率分别平均提高了 7.7 倍和 2.0 倍，同时在精于MMR的细胞类型中将编辑/插入删除比率提高了 3.4 倍。在预期编辑附近战略性地建立沉默突变可以通过规避 MMR 来增强引导编辑结果。 引导编辑器蛋白质优化导致 PEmax 架构在 HeLa 细胞中将编辑效率平均提高 2.8 倍。

这些发现丰富了人们对引导编辑的理解，并建立了引导编辑系统，该系统在 7 种哺乳动物细胞类型的 191 次编辑中显示出实质性改进。

据了解，虽然引导编辑能够在 DNA 中实现精确的序列变化，但引导编辑的细胞决定因素仍然知之甚少。

附：英文原文

Title: Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Author: 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

Issue&Volume: 2021-10-14

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.

DOI: 10.1016/j.cell.2021.09.018

Source: https://www.cell.com/cell/fulltext/S0092-8674(21)01065-5