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Related Experiment Video

Updated: Jun 7, 2026

Breeding by Design for Functional Rice with Genome Editing Technologies
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Breeding by Design for Functional Rice with Genome Editing Technologies

Published on: January 3, 2025

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

Sujie Zhang1, Jingqi Du1, Guigen Ma2

  • 1State 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.

Journal of Advanced Research
|June 5, 2026
PubMed
Summary

This study optimized prime editing for efficient large DNA fragment replacement in rice. An engineered reverse transcriptase variant significantly enhanced editing efficiency, enabling precise gene rewriting for crop improvement.

Keywords:
Gene correctionIsogenic sequence replacementM−MLV reverse transcriptaseOryza sativaL.Prime editing

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Area of Science:

  • Plant biotechnology
  • Genome editing
  • Molecular biology

Background:

  • Large genomic fragment replacement is challenging in plant genome editing.
  • This limits the use of natural genetic diversity for crop trait improvement.

Purpose of the Study:

  • To develop an optimized prime editing (PE) strategy for high-efficiency, large-fragment isogenic sequence replacement (ISR) in rice.
  • To engineer a novel reverse transcriptase variant for enhanced editing.

Main Methods:

  • Systematic comparison of nuclease-based PE, template-jumping PE (TJ-PE), and GRAND PE strategies.
  • Engineering of Moloney murine leukemia virus reverse transcriptase (RT) variants.
  • Performance evaluation in rice protoplasts and transgenic lines.

Main Results:

  • Template-jumping PE (TJ-PE) demonstrated superior ISR efficiency and precision.
  • Engineered RT variant rPE14e4 enhanced ISR efficiency 4.5-fold, enabling precise replacements up to 250 bp.
  • Disrupting microhomology between primer binding site (PBS) and reverse transcription template (RTT) improved editing fidelity.
  • A 174-bp coding region of the xa10 gene was precisely rewritten in rice.

Conclusions:

  • An efficient PE-mediated system for large-fragment ISR in rice was established.
  • The optimized strategy and engineered RT variant enhance precise gene rewriting capabilities.
  • This accelerates functional genomics and molecular breeding in crops.