期刊文献

Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA 收藏

使用RNA-POL-II驱动的工程Pegrna扩大Prime编辑工具包

原文求助发布源

作者

作者单位

[1]School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.[2]Zhejiang Lab, Hangzhou, Zhejiang 311121, China, Zhejiang Lab, Hangzhou, Zhejiang 311121, China.[3]University of Chinese Academy of Sciences, Beijing 100049, China, University of Chinese Academy of Sciences, Beijing 100049, China.[4]Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100 Shaanxi, China, Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100 Shaanxi, China.[5]National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran, National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran[6]Oujiang Laboratory, Wenzhou, Zhejiang, China, Key Laboratory of Alzheimer's Disease of Zhejiang Province, Institute of Aging, Wenzhou Medical University; Oujiang Laboratory, Wenzhou, Zhejiang, China[7]Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China, Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou 310029, China[8]8These authors contributed equally

关键词

CRISPR;prime editor;human pluripotent stem cells;p53 response;

关键词译文

CRISPR; Prime Editor;人类多能干细胞； p53响应；

发布日期

7 September 2022

页码

2923-2932

DOI

10.1016/j.ymthe.2022.07.002

来源信息

Molecular Therapy ISSN：1525-0016, 2022年, 30卷, 9期, 2923-2932页

摘要

The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.

摘要译文

Prime编辑器是用于针对性精确编辑的多功能工具，以生成点突变，小插入或真核生物中的小缺失。但是，规范PE3系统的效率较低，特别是在原代细胞或多能干细胞中。在这里，我们采用了RNA聚合酶II启动子，而不是RNA聚合酶III启动子（其应用受特定DNA环境的限制）来生产CSY4所处理的内含子质量编辑指南RNA（PEGRNA），并与其他优化一起实现了有效的靶标（t） - 含有的pegrnas，以及组合和条件遗传编辑。我们还发现，同时抑制DNA不匹配修复和DNA损伤响应可以在人类胚胎干细胞中获得有效而准确的编辑。这些发现减轻了基于RNA聚合酶III（RNA-POL-III）的基础编辑器的限制，并扩大了Prime编辑的应用。

Shisheng Huang[1][2][3][8];Zhenwu Zhang[1][8];Wanyu Tao[1][3][8];Yao Liu[4];Xiangyang Li[1][3];Xiaolong Wang[4];Javad Harati[5];Peng-Yuan Wang[6];Xingxu Huang[1][2][7]PersonEnvelope;Chao-Po Lin[1]PersonEnvelope. Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA[J]. Molecular Therapy, 2022,30(9): 2923-2932