Xiong Youcai, Su Yinyu, He Ruigao, Han Xiaosong, Li Sheng, Liu Minghuan, Xi Xiaoning, Liu Zijia, Wang Heng, Xie Shengsong, Xu Xuewen, Li Kui, Zhang Jifeng, Xu Jie, Li Xinyun, Zhao Shuhong, Ruan Jinxue
Frontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural University, Wuhan, PR China.
Breeding and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, PR China.
Nat Commun. 2025 Feb 13;16(1):1592. doi: 10.1038/s41467-025-56734-9.
Prime editing systems (PEs) hold great promise in modern biotechnology. However, their editing range is limited as PEs can only modify the downstream sequences of the pegRNA nick. Here, we report the development of the extended prime editor system (EXPERT) to overcome this limitation by using an extended pegRNA (ext-pegRNA) with modified 3' extension, and an additional sgRNA (ups-sgRNA) targeting the upstream region of the ext-pegRNA. We demonstrate that EXPERT can efficiently perform editing on both sides of the ext-pegRNA nick, a task that is unattainable by canonical PEs. EXPERT exhibits prominent capacity in replacing sequences up to 88 base pairs and inserting sequences up to 100 base pairs within the upstream region of the ext-pegRNA nick. Compared to canonical PEs such as PE2, the utilization of the EXPERT strategy significantly enhances the editing efficiency for large fragment edits with an average improvement of 3.12-fold, up to 122.1 times higher. Safety wise, the use of ups-sgRNA does not increase the rates of undesirable insertions and deletions (indels), as the two nicks are on the same strand. Moreover, we do not observe increased off-target editing rates genome-wide. Our work introduces EXPERT as a PE tool with significant potential in life sciences.
引导编辑系统（PEs）在现代生物技术中具有巨大潜力。然而，它们的编辑范围有限，因为PEs只能修饰pegRNA切口下游的序列。在此，我们报告了扩展引导编辑器系统（EXPERT）的开发，以克服这一限制，该系统使用具有修饰3'端延伸的扩展pegRNA（ext-pegRNA），以及靶向ext-pegRNA上游区域的额外sgRNA（ups-sgRNA）。我们证明，EXPERT可以在ext-pegRNA切口的两侧高效地进行编辑，这是传统PEs无法完成的任务。EXPERT在ext-pegRNA切口上游区域替换长达88个碱基对的序列和插入长达100个碱基对的序列方面表现出显著能力。与诸如PE2等传统PEs相比，EXPERT策略的使用显著提高了大片段编辑的效率，平均提高了3.12倍，最高可达122.1倍。在安全性方面，使用ups-sgRNA不会增加意外插入和缺失（indels）的发生率，因为两个切口在同一条链上。此外，我们在全基因组范围内未观察到脱靶编辑率增加。我们的工作将EXPERT作为一种在生命科学中具有巨大潜力的PE工具引入。
