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

Jun 5, 2026Journal of advanced research

Precise replacement of large DNA sections in rice using improved prime editing enzymes

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Abstract

TJ-PE enhanced isogenic sequence replacement efficiency by 4.5-fold compared to other strategies.

An engineered reverse transcriptase variant, rPE14e4, was identified as crucial for improving ISR efficiency.
Precise replacements of up to 250 bp were achieved using the optimized rPE14e4-TJ-PE system.
Unintended microhomology between the primer binding site and reverse transcription template was linked to on-target byproducts.
Disrupting this microhomology improved the fidelity of the editing process.
A successful rewrite of a 174-bp coding region of the xa10 gene was demonstrated in rice.

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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.

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

Abstract

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