Efficient large-fragment isogenic sequence replacement in rice via prime editing with engineered reverse transcriptase variants

Sujie Zhang; Jingqi Du; Guigen Ma; Chao Yang; Bin Ren; Fang Yan; Shaofang Li; Xueping Zhou; Huanbin Zhou

doi:10.1016/j.jare.2026.06.006

Efficient large-fragment isogenic sequence replacement in rice via prime editing with engineered reverse transcriptase variants

J Adv Res. 2026 Jun 4:S2090-1232(26)00459-5. doi: 10.1016/j.jare.2026.06.006. Online ahead of print.

Authors

Sujie Zhang¹, Jingqi Du¹, Guigen Ma², Chao Yang³, Bin Ren¹, Fang Yan², Shaofang Li⁴, Xueping Zhou⁵, Huanbin Zhou⁶

Affiliations

¹ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Scientific Observing and Experimental Station of Crop Pests in Guilin, Ministry of Agriculture and Rural Affairs, Guilin 541399, China.
² State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572024, China.
³ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
⁴ State Key Laboratory of Vegetable Biobreeding, National Engineering Research Center for Vegetables, Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Science, Beijing 100097, China.
⁵ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China.
⁶ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; Scientific Observing and Experimental Station of Crop Pests in Guilin, Ministry of Agriculture and Rural Affairs, Guilin 541399, China; Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572024, China. Electronic address: zhouhuanbin@caas.cn.

PMID: 42248511
DOI: 10.1016/j.jare.2026.06.006

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.

Keywords: Gene correction; Isogenic sequence replacement; M−MLV reverse transcriptase; Oryza sativaL.; Prime editing.