EXPERT expands prime editing efficiency and range of large fragment edits

Youcai Xiong, Yinyu Su, Ruigao He, Xiaosong Han, Sheng Li, Minghuan Liu, Xiaoning Xi, Zijia Liu, Heng Wang, Shengsong Xie, Xuewen Xu, Kui Li, Jifeng Zhang, Jie Xu, Xinyun Li (), Shuhong Zhao () and Jinxue Ruan ()
Additional contact information
Youcai Xiong: Huazhong Agricultural University
Yinyu Su: Huazhong Agricultural University
Ruigao He: Huazhong Agricultural University
Xiaosong Han: Huazhong Agricultural University
Sheng Li: Huazhong Agricultural University
Minghuan Liu: Huazhong Agricultural University
Xiaoning Xi: Huazhong Agricultural University
Zijia Liu: Huazhong Agricultural University
Heng Wang: Huazhong Agricultural University
Shengsong Xie: Huazhong Agricultural University
Xuewen Xu: Huazhong Agricultural University
Kui Li: Chinese Academy of Agricultural Sciences
Jifeng Zhang: University of Michigan Medical School
Jie Xu: University of Michigan Medical School
Xinyun Li: Huazhong Agricultural University
Shuhong Zhao: Huazhong Agricultural University
Jinxue Ruan: Huazhong Agricultural University

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-56734-9

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