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Multiplexed, precise genome engineering in monocots with twin prime editing systems.

Hongchao Li1, Zhuangzhuang Chai1, Xiaoli Shi1

  • 1New Cornerstone Science Laboratory, Laboratory of Advanced Breeding Technologies, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

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|June 5, 2026
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Summary

A new twin prime editing-based knockout (TKO) system precisely disables genes in crops like rice, maize, and wheat. This advanced genome engineering tool achieves high knockout efficiencies and enables simultaneous editing of multiple genes.

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Area of Science:

  • Plant science
  • Genomics
  • Biotechnology

Background:

  • Simultaneously introducing multiple genomic edits in crops is a significant challenge in genome engineering.
  • Existing methods often result in undesirable in-frame mutations, complicating gene knockout.

Purpose of the Study:

  • To develop a novel system for precise, simultaneous gene knockout in crop plants.
  • To improve knockout efficiency and minimize off-target mutations compared to existing technologies.

Main Methods:

  • Development of a twin prime editing-based knockout (TKO) system utilizing stop codon clusters (SCCs).
  • Testing TKO efficiency in rice, maize, and wheat protoplasts and regenerated rice plants.
  • Comparison of TKO performance against Cas9 in hexaploid wheat for triple-homolog knockouts.
  • Development of orthogonal TKO editors for simultaneous knockout of multiple genes.
  • Integration of TKO with prime editing to create TRIM1 for simultaneous knockout and precise editing.
  • Extension of capabilities with TRIM2 for kilobase-scale modifications via a prime editor-recombinase system.

Main Results:

  • TKO achieved high knockout efficiencies: up to 70.5% in rice, 58.6% in maize, and 75.1% in wheat protoplasts.
  • Heritable knockout alleles were produced in 96.8% of regenerated rice plants.
  • TKO outperformed Cas9 4.2-fold in generating triple-homolog knockouts in wheat, with fewer in-frame mutations.
  • Orthogonal TKO editors enabled simultaneous knockout of up to ten genes without cross-interference.
  • TRIM1 achieved 22.8% coediting of four genes in rice.
  • TRIM2 enabled a 4.9-kb insertion (1.2% efficiency) and gene knockout (up to 79.8%) in protoplasts.

Conclusions:

  • The TKO system offers a precise and efficient method for gene knockout in major crop species.
  • TKO significantly improves multiplex gene editing capabilities, outperforming Cas9 in specific applications.
  • TRIM systems expand genome engineering toolkits for simultaneous knockout and complex modifications, advancing crop improvement.