Enhanced prime editing systems by manipulating cellular determinants of editing outcomes,Cell

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Enhanced prime editing systems by manipulating cellular determinants of editing outcomes
Cell ( IF 45.5 ) Pub Date : 2021-10-14 , DOI: 10.1016/j.cell.2021.09.018
Peter J Chen ₁ , Jeffrey A Hussmann ₂ , Jun Yan ₃ , Friederike Knipping ₄ , Purnima Ravisankar ₅ , Pin-Fang Chen ₆ , Cidi Chen ₆ , James W Nelson ₁ , Gregory A Newby ₁ , Mustafa Sahin ₇ , Mark J Osborn ₄ , Jonathan S Weissman ₈ , Britt Adamson ₉ , David R Liu ₁

Affiliation

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Department of Pediatrics, University of Minnesota, Minneapolis, MN 55454, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55108, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA.
Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.

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.

中文翻译：

通过操纵编辑结果的细胞决定因素来增强原始编辑系统

虽然初始编辑能够在 DNA 中实现精确的序列变化，但对初始编辑的细胞决定因素仍然知之甚少。使用合并的 CRISPRi 筛选，我们发现 DNA 错配修复 (MMR) 会阻碍原始编辑并促进不需要的插入缺失副产物。我们开发了 PE4 和 PE5 初始编辑系统，其中工程化 MMR 抑制蛋白的瞬时表达与 PE2 和 PE3 系统相比，将替换、小插入和小删除初始编辑的效率平均提高了 7.7 倍和 2.0 倍，分别地，同时在 MMR 熟练的细胞类型中将编辑/插入缺失比率提高 3.4 倍。在预期编辑附近战略性地安装沉默突变可以通过规避 MMR 来增强主要编辑结果。Prime 编辑器蛋白质优化产生了一种 PEmax 架构，可将编辑效率提高 2 倍。HeLa 细胞中平均为 8 倍。这些发现丰富了我们对原始编辑的理解，并建立了原始编辑系统，在七种哺乳动物细胞类型的 191 次编辑中显示出显着改善。

更新日期：2021-10-28

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