Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity
Prime editing (PE) is a precise tool for introducing genetic mutations in eukaryotes. Extending the efficient editing scope and mitigating undesired byproducts are possible. We introduce reverse PE (rPE), a SpCas9-directed variant that enabled DNA editing at the 3' direction of HNH-mediated nick site. The rPE leveraging nCas9-D10A and rPE gRNA targeting the 5' direction of HNH-mediated nick site inscribes genetic alterations, achieving a reverse editing window and potentially high fidelity. HNH and reverse transcriptase engineered using protein language models in conjunction with La facilitate circular erPEmax and erPE7max, achieving editing efficiency up to 44.41% without nick gRNA or positive selection. Furthermore, our findings underscore the capability of rPE in inserting functionally enhanced variant (PIK3CD<sup>E527G</sup>) for cell therapy. By expanding the editing scope and enhancing genomic manipulability, rPE represents a meaningful advancement in prime editing, improving its utility for research and therapeutic applications.
Authors
- Chongbiao Huang (ORCID: https://orcid.org/0000-0001-9468-9168)
- Lei Shi (ORCID: https://orcid.org/0000-0003-1679-3618)
- Jie Deng (ORCID: https://orcid.org/0000-0001-9774-1240)
- Jun Yu (ORCID: https://orcid.org/0000-0003-3435-6550)
- Xiuchao Wang (ORCID: https://orcid.org/0000-0002-3205-8544)
- Qingxiao Fang
- Keshan Wang (ORCID: https://orcid.org/0000-0002-4128-2214)
- Changhao Bi (ORCID: https://orcid.org/0000-0002-1940-8511)
- Jihui Hao (ORCID: https://orcid.org/0000-0002-1607-1730)
- Xiaoping Zhang (ORCID: https://orcid.org/0000-0001-5428-4545)
- Jin Zhang (ORCID: https://orcid.org/0000-0002-8397-5078)
- Rui Li (ORCID: https://orcid.org/0000-0002-2379-5219)
- Mengyu Li (ORCID: https://orcid.org/0000-0002-3122-650X)
- Yukuan Feng
- Chao Yang (ORCID: https://orcid.org/0000-0003-3422-2763)
- Xueli Zhang (ORCID: https://orcid.org/0000-0002-8660-4037)
- Tianxing Zhou (ORCID: https://orcid.org/0009-0006-0150-1343)
Institutions
- Union Hospital (HK)
- Tianjin University of Science and Technology (CN)
- Nankai University (CN)
- National Medical Products Administration (CN)
- Tianjin Medical University Cancer Institute and Hospital (CN)
- Tianjin Institute of Pharmaceutical Research (China) (CN)
- Ministry of Education (PT)
- Tianjin Institute of Industrial Biotechnology (CN)
- Huazhong University of Science and Technology (CN)
- Tianjin Medical University (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-06-03
- DOI
- https://doi.org/10.1038/s41467-025-60495-w
- Citations
- 5
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 4.03
Funders
- National Natural Science Foundation of China
- Natural Science Foundation of Tianjin City
- Tianjin Medical University
- National Key Research and Development Program of China