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EXPERT expands prime editing efficiency and range of large fragment edits

Author

Listed:
  • Youcai Xiong

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Yinyu Su

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Ruigao He

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Xiaosong Han

    (Huazhong Agricultural University
    Huazhong Agricultural University
    Yazhouwan National Laboratory)

  • Sheng Li

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Minghuan Liu

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Xiaoning Xi

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Zijia Liu

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Heng Wang

    (Huazhong Agricultural University
    Huazhong Agricultural University)

  • Shengsong Xie

    (Huazhong Agricultural University
    Huazhong Agricultural University
    Huazhong Agricultural University
    Huazhong Agricultural University)

  • Xuewen Xu

    (Huazhong Agricultural University
    Huazhong Agricultural University
    Huazhong Agricultural University
    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
    Huazhong Agricultural University
    Huazhong Agricultural University
    Huazhong Agricultural University)

  • Shuhong Zhao

    (Huazhong Agricultural University
    Huazhong Agricultural University
    Yazhouwan National Laboratory
    Huazhong Agricultural University)

  • Jinxue Ruan

    (Huazhong Agricultural University
    Huazhong Agricultural University)

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.

Suggested Citation

  • 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 Zh, 2025. "EXPERT expands prime editing efficiency and range of large fragment edits," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-56734-9
    DOI: 10.1038/s41467-025-56734-9
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    References listed on IDEAS

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