Advanced science (Weinheim, Baden-Wurttemberg, Germany)Jun 26, 2026

Finding and Improving a Rat Virus Enzyme for More Efficient Prime Editing

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Nineteen novel active reverse transcriptases were identified from a screening of 558 candidates, with enRERV-RT showing the highest activity.

The optimized enRERV-RT variant outperforms conventional M-MLV-RT-based systems by 1.20-fold in mammalian and plant cells.
At hard-to-edit loci, enRERV-RT provides a 1.88-fold increase in editing efficiency compared to M-MLV-RT.
The high-throughput platform TRAP-seq-PE was developed to evaluate prime editor performance systematically.
PE systems utilizing enRERV-RT demonstrate higher editing efficiencies across various mutation types than those using M-MLV-RT.
The advancements may facilitate improvements in clinical gene therapy and crop breeding.

Simplified

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.