Abstract

The development of efficient and precise genome-editing tools is crucial for advancing functional genomics and improving crops. Our previously established nuclease-mediated prime editing (NM-PE) system, which combines the SpCas9 nuclease with prime editing based on microhomology-mediated end joining, enables the seamless insertion of small DNA fragments into plant genomes to add tags to genes of interest. However, the efficiency of 3×FLAG sequence insertion via NM-PE requires further improvement. Here, we report a significant optimization of this system by replacing the M-MLV reverse transcriptase (RT) with evolved variants of the retrotransposon RT Tf1 derived from the mammalian PE6 system. Through codon optimization, we generated the evoTf1M4 variant, which substantially enhanced the efficiency of NM-PE. The optimized construct rPE20aV3 achieved up to 18.75% precise insertion of a 66-bp 3×FLAG sequence at endogenous loci, representing a three-fold improvement over the original NM-PE system. Our results demonstrate that Tf1-aided optimization of NM-PE serves as an efficient platform for seamless insertion of a 3×FLAG sequence in rice, offering broad potential for advanced genome engineering in plants.