@article{97d5a829090b4997be74657552fc4b42,
title = "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes",
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.",
keywords = "CRISPR-Cas9, Repair-seq, genome editing, mismatch repair, prime editing",
author = "Chen, {Peter J.} and Hussmann, {Jeffrey A.} and Jun Yan and Friederike Knipping and Purnima Ravisankar and Chen, {Pin Fang} and Cidi Chen and Nelson, {James W.} and Newby, {Gregory A.} and Mustafa Sahin and Osborn, {Mark J.} and Weissman, {Jonathan S.} and Britt Adamson and Liu, {David R.}",
note = "Funding Information: This work was supported by the Merkin Institute of Transformative Technologies in Healthcare; NIH grants U01AI142756, RM1HG009490, R01EB031172, and R35GM118062; the Howard Hughes Medical Institute; the Loulou Foundation; and the Bill & Melinda Gates Foundation. P.J.C. was supported by the NSF Graduate Research Fellowship. J.A.H. was the Rebecca Ridley Kry Fellow of the Damon Runyon Cancer Research Foundation (DRG-2262-16). J.Y. was supported by a fellowship provided by the China Scholarship Council (CSC), based on the April 2015 Memorandum of Understanding between the CSC and Princeton University. J.W.N. was supported by a Jane Coffin Childs postdoctoral fellowship. G.A.N. was supported by a Helen Hay Whitney postdoctoral fellowship. M.J.O. was supported by a Saint Baldrick's Foundation Scholar Award, the Kidz1stFund, and the Chambers Family Innovation Fund. Work in the lab of B.A. was supported by NIH grant R35GM138167-01, Rutgers Cancer Institute of New Jersey via NCI-CCSG grant P30CA072720), and the Searle Scholars Program. We thank A. Anzalone, K. Everette, J. Davis, L. Koblan, A. Le, and D. Simpson for helpful discussions and technical advice; Dr. Anahita Vieira for assistance editing the manuscript; the Princeton University Flow Cytometry Resource Facility, which is supported by NCI-CCSG grant P30CA072720-5921; Wei Wang and the Genomics Core Facility in the Lewis-Sigler Institute for Integrative Genomics at Princeton; and the Human Neuron Core at Boston Children's Hospital, which is supported by NIH grant U54HD090255. P.J.C. J.A.H. B.A. and D.R.L. designed the research and wrote the manuscript. P.J.C. J.Y. P.R. and B.A. performed Repair-seq screens. J.A.H. analyzed Repair-seq screen data. P.J.C. developed PE4, PE5, and PEmax. P.J.C. and J.A.H. analyzed sequencing data. J.Y. performed K562 and U2OS cell experiments. F.K. and M.J.O. performed T cell experiments with input from G.A.N. P.-F.C. and C.C. performed iPSC experiments with assistance from P.J.C. and J.W.N. M.S. M.J.O. J.S.W. B.A. and D.R.L. supervised the project. P.J.C. J.A.H. B.A. and D.R.L. have filed patent applications on aspects of this work through their respective institutions. J.A.H. is a consultant for Tessera Therapeutics. P.-F.C. is currently an employee of Tessera Therapeutics. B.A. was a member of a ThinkLab Advisory Board for, and holds equity in, Celsius Therapeutics. D.R.L. is a consultant and equity holder of Beam Therapeutics, Prime Medicine, Pairwise Plants, and Chroma Medicine. Funding Information: This work was supported by the Merkin Institute of Transformative Technologies in Healthcare ; NIH grants U01AI142756 , RM1HG009490 , R01EB031172 , and R35GM118062 ; the Howard Hughes Medical Institute ; the Loulou Foundation ; and the Bill & Melinda Gates Foundation . P.J.C. was supported by the NSF Graduate Research Fellowship. J.A.H. was the Rebecca Ridley Kry Fellow of the Damon Runyon Cancer Research Foundation ( DRG-2262-16 ). J.Y. was supported by a fellowship provided by the China Scholarship Council (CSC), based on the April 2015 Memorandum of Understanding between the CSC and Princeton University. J.W.N. was supported by a Jane Coffin Childs postdoctoral fellowship. G.A.N. was supported by a Helen Hay Whitney postdoctoral fellowship. M.J.O. was supported by a Saint Baldrick's Foundation Scholar Award, the Kidz1stFund, and the Chambers Family Innovation Fund. Work in the lab of B.A. was supported by NIH grant R35GM138167-01 , Rutgers Cancer Institute of New Jersey via NCI-CCSG grant P30CA072720 ), and the Searle Scholars Program . We thank A. Anzalone, K. Everette, J. Davis, L. Koblan, A. Le, and D. Simpson for helpful discussions and technical advice; Dr. Anahita Vieira for assistance editing the manuscript; the Princeton University Flow Cytometry Resource Facility, which is supported by NCI-CCSG grant P30CA072720-5921 ; Wei Wang and the Genomics Core Facility in the Lewis-Sigler Institute for Integrative Genomics at Princeton ; and the Human Neuron Core at Boston Children{\textquoteright}s Hospital, which is supported by NIH grant U54HD090255 . Publisher Copyright: {\textcopyright} 2021 The Author(s)",
year = "2021",
month = oct,
day = "28",
doi = "10.1016/j.cell.2021.09.018",
language = "English (US)",
volume = "184",
pages = "5635--5652.e29",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "22",
}