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