Abstract

INTRODUCTION Achieving efficient and precise replacement of large genomic fragments with isogenic sequences remains a major challenge in plant genome editing, limiting the exploitation of natural allelic diversity for trait improvement. OBJECTIVES This study aims to develop an optimized prime editing (PE) strategy for high-efficiency, large-fragment isogenic sequence replacement (ISR) in rice. METHODS We systematically compared nuclease-based PE, template-jumping PE (TJ-PE), and GRAND PE strategies. We engineered a series of M-MLV reverse transcriptase (RT) variants and evaluated their performance in rice protoplasts and stable transgenic lines. RESULTS TJ-PE outperformed other strategies in ISR efficiency and precision. Engineering the Moloney murine leukemia virus reverse transcriptase yielded rPE14e4 (T128N/D200C/V223Y/L435K), which enhanced ISR efficiency by 4.5-fold and enabled precise replacements up to 250 bp. We also discovered that unintended microhomology between primer binding site (PBS) and reverse transcription template (RTT) can cause on-target byproducts, and its disruption improves editing fidelity. Applying the optimized rPE14e4-TJ-PE system, we successfully rewrote a 174-bp coding region of the xa10 gene in the elite rice cultivar N9208. CONCLUSION We established an efficient PE-mediated system for large-fragment ISR in rice. The optimized strategy and engineered RT variant significantly expand the capability for precise gene rewriting, accelerating functional genomics and molecular breeding in crops.