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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\u2010RT, derived from\n                    <jats:italic>Rattus norvegicus<\/jats:italic>\n                    , exhibited the highest activity. Through structure\u2010guided engineering and deep mutational scanning, we developed an optimized variant, enRERV\u2010RT, which outperforms conventional M\u2010MLV\u2010RT\u2010based PE systems by 1.20\u2010fold in mammalian and plant cells, and by 1.88\u2010fold at hard\u2010to\u2010edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high\u2010throughput platform, TRAP\u2010seq\u2010PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV\u2010RT exhibited higher editing efficiencies than those based on M\u2010MLV\u2010RT. Collectively, our work establishes a versatile, high\u2010efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.\n                  <\/jats:p>","DOI":"10.1002\/advs.75888","type":"journal-article","created":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T12:31:41Z","timestamp":1782477101000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing"],"prefix":"10.1002","volume":"13","author":[{"given":"Linsha","family":"Ma","sequence":"first","affiliation":[{"name":"Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops Key Laboratory for Enhancing Resource Use Efficiency of Crops in South China State Key Laboratory For Conservation and Utilization of Subtropical Agro\u2010Bioresources Guangdong Provincial Key Laboratory of Plant Molecular 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