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
Abstract
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.