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Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing

Year: 2026
Type: article
Abstract: 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.
DOI: 10.1002/advs.75888
Authors: Linsha Ma, Pengcheng Yao, Shengqi Wu, Yuanyuan Shi, Lang Qin, Baitao Li, Jiayi Zhu, Minhua Huang, Yichong Zhu, Yuwen Song, Jinhuan Pang, Ziping Guo, Guochuan Wu, Chen Wang, Kewei Xu, Ruihua Huang, Quan Kuang, Liang Qu, Changtian Pan, Xianrong Xie, Qinlong Zhu, Jiaying Huang, Qiupeng Lin
Institutions: South China Agricultural University, Ministry of Agriculture and Rural Affairs, State Key Laboratory For Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou Institutes of Biomedicine and Health, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, Guangzhou Medical University, Southwest University, Beijing Normal-Hong Kong Baptist University, Southern Medical University, South China Normal University, Chinese Academy of Sciences, Shenzhen Institutes of Advanced Technology, Shenzhen Institute of Synthetic Biology, Nanchang Normal University, Shanghai Medical College of Fudan University, Fudan University, Zhongshan Hospital, Zhejiang University
Language: English
Open Access status: gold
Estimated APC paid: $6,730
Awards: G1813156610, G231272093, G2818190089, G3852078645, G4631792171, G5938979750, G8693854471
OpenAlex ID: W7166116586
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Cites: 42
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