Establishment of a novel prime editing system by introducing the reverse transcriptase Tf1 combined with high-temperature treatment in rice

<p indent="0mm">Reverse transcriptase is a vital component of the prime editing (PE) system and responsible for converting RNA templates carrying mutational information into DNA, thereby achieving precise editing of target genes. In plants, the PE system has demonstrated powerful precision editing capabilities; however, there are relatively limited resources for effective reverse transcriptases, with current reliance primarily on the optimization and application of Moloney murine leukemia virus reverse transcriptase (M-MLV). The aim of this study was to enhance the performance of the plant PE system by introducing novel reverse transcriptases based on the highly efficient plant ePE2 system. First, the reverse transcriptase Tf1 (from the <italic>Schizosaccharomyces pombe</italic> Tf1 retrotransposon) has demonstrated high stability and efficiency after phage-assisted evolution in mammalian cells, suggesting that it may possess high reverse transcription capabilities. Therefore, we replaced the original M-MLV reverse transcriptase with the Tf1 reverse transcriptase after rice codon optimization; this strategy is referred to as 6c. Second, a nCas9 variant (K775R and K918A) has shown a role in stabilizing the R-loop and mediating conformational changes in the HNH domain upon DNA binding, significantly enhancing editing efficiency in mammalian cells. Thus, we introduced the same two-point mutations, K775R and K918A, into the ePE2 system; this strategy is referred to as 6e. Finally, the 6c and 6e strategies were combined to develop the novel ePE2-6ce system. For validating the feasibility and efficiency of the newly constructed ePE2-6ce system, we selected three specific targets, <italic>OsCDC48</italic>, <italic>OsALS</italic>, and <italic>OsROC5</italic>, in the rice genome for verification. These three sites represent different editing types and can comprehensively evaluate the editing capability of the ePE2-6ce system. The experimental results demonstrated that in the resistant callus, the ePE2-6ce system achieved editing efficiencies of 6.82%, 34.55%, and 23.72% for the <italic>OsCDC48</italic>, <italic>OsALS</italic>, and <italic>OsROC5 </italic>sites, respectively, whereas in the regenerated T₀ generation plants, the corresponding editing efficiencies were 22.00%, 56.52%, and 58.00%. These data demonstrate the powerful precision editing capability of the ePE2-6ce system. Remarkably, appropriate high-temperature treatment during the callus screening stage can improve the activity of the reverse transcriptase. Therefore, we verified the effect of high-temperature treatment on the editing efficiency of the ePE2-6ce system. The results demonstrated that when combined with high-temperature treatment, the editing efficiency of the <italic>OsROC5</italic> site increased by 1.97- and 1.12-fold in the callus and T₀ generation plants, respectively. This result provides novel insights into further improving the editing efficiency of the ePE2-6ce system through environmental factors. To summarize, we successfully applied the novel reverse transcriptase Tf1 to precise gene editing in rice, expanding the selection range of reverse transcriptases in plant PE systems. The ePE2-6ce system provides a powerful new tool for crop genetic improvement and functional genomics research, while also laying an important foundation for the optimization and application of reverse transcriptases in future plant PE systems.