EXPERT expands prime editing efficiency and range of large fragment edits
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 usi...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-02-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-56734-9 |
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| author | 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 Jinxue Ruan |
| author_facet | 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 Jinxue Ruan |
| author_sort | Youcai Xiong |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-0189db170ad8440e9c1c6f05c24541cf |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-0189db170ad8440e9c1c6f05c24541cf2025-08-20T03:00:59ZengNature PortfolioNature Communications2041-17232025-02-0116111310.1038/s41467-025-56734-9EXPERT expands prime editing efficiency and range of large fragment editsYoucai Xiong0Yinyu Su1Ruigao He2Xiaosong Han3Sheng Li4Minghuan Liu5Xiaoning Xi6Zijia Liu7Heng Wang8Shengsong Xie9Xuewen Xu10Kui Li11Jifeng Zhang12Jie Xu13Xinyun Li14Shuhong Zhao15Jinxue Ruan16Frontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityAgricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural SciencesCenter for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical SchoolCenter for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical SchoolFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityFrontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural UniversityAbstract 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.https://doi.org/10.1038/s41467-025-56734-9 |
| spellingShingle | 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 Jinxue Ruan EXPERT expands prime editing efficiency and range of large fragment edits Nature Communications |
| title | EXPERT expands prime editing efficiency and range of large fragment edits |
| title_full | EXPERT expands prime editing efficiency and range of large fragment edits |
| title_fullStr | EXPERT expands prime editing efficiency and range of large fragment edits |
| title_full_unstemmed | EXPERT expands prime editing efficiency and range of large fragment edits |
| title_short | EXPERT expands prime editing efficiency and range of large fragment edits |
| title_sort | expert expands prime editing efficiency and range of large fragment edits |
| url | https://doi.org/10.1038/s41467-025-56734-9 |
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