Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing
CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.
Authors
- Y F Shi
- S S Wu
- Changtian Pan (ORCID: https://orcid.org/0000-0002-5852-059X)
- Qiupeng Lin (ORCID: https://orcid.org/0000-0002-7944-6683)
- Qinlong Zhu (ORCID: https://orcid.org/0000-0001-7622-6450)
- Xianrong Xie (ORCID: https://orcid.org/0000-0002-1438-7766)
- Chen Wang (ORCID: https://orcid.org/0000-0001-7857-5435)
- Ke Xu (ORCID: https://orcid.org/0000-0001-8669-3909)
- Jinhuan Pang (ORCID: https://orcid.org/0000-0002-6569-028X)
- Jiayi Zhu (ORCID: https://orcid.org/0009-0003-0463-9442)
- Pengcheng Yao (ORCID: https://orcid.org/0009-0001-0874-7577)
- Lang Qin (ORCID: https://orcid.org/0009-0008-0284-1264)
- Li B
- Linsha Ma
- Guochuan Wu
- Jiaying Huang
- Y Song
- Liang Qu
- Yichong Zhu
- Ruihua Huang
- Minhua Huang
- Ziping Guo
- Quan Kuang
Institutions
- South China Agricultural University (CN)
- Beijing Normal-Hong Kong Baptist University (CN)
- Zhejiang A & F University (CN)
- Shanghai Medical College of Fudan University (CN)
- Hong Kong Baptist University (HK)
- Southwest University (CN)
- South China Normal University (CN)
- Fudan University (CN)
- First Affiliated Hospital of Guangzhou Medical University (CN)
- Nanchang Normal University (CN)
- Ministry of Agriculture (ID)
- Panyu District Central Hospital (CN)
- Zhongshan Hospital (CN)
- Shenzhen Institutes of Advanced Technology (CN)
- State Key Laboratory of Respiratory Disease (CN)
- Southern Medical University (CN)
- Guangzhou Medical University (CN)
Publication Details
- Journal
- Advanced Science
- Published
- 2026-06-26
- DOI
- https://doi.org/10.1002/advs.75888
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Natural Science Foundation of China