Development of a versatile nuclease prime editor with upgraded precision.
Xiangyang Li, Guiquan Zhang, Shisheng Huang, Yao Liu, Jin Tang, Mingtian Zhong, Xin Wang, Wenjun Sun, Yuan Yao, Quanjiang Ji, Xiaolong Wang, Jianghuai Liu, Shiqiang Zhu, Xingxu Huang
Author Information
Xiangyang Li: Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China.
Yao Liu: Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China.
Jin Tang: Zhejiang Lab, Hangzhou, Zhejiang, 311121, China.
Mingtian Zhong: Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, 510631, China.
Xin Wang: Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China.
Wenjun Sun: Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China.
Yuan Yao: ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 311215, China.
Quanjiang Ji: School of Physical Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China. ORCID
Xiaolong Wang: Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China. ORCID
Jianghuai Liu: State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center at Medical School of Nanjing University, 210061, Nanjing, China. liujianghuai@nju.edu.cn. ORCID
Xingxu Huang: Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China. huangxx@shanghaitech.edu.cn. ORCID
The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential.
References
Annu Rev Biochem. 2010;79:181-211
[PMID: 20192759]
DNA Repair (Amst). 2008 Oct 1;7(10):1765-71
[PMID: 18675941]
