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Engineering a robust Cas12i3 variant-mediated wheat genome editing system
Wenxue Wang1,2, Lei Yan1, Jingying Li1,2
1State Key Laboratory of Crop Gene Resources and Breeding, Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
Plant Biotechnology Journal
|December 18, 2024
Summary
Researchers developed a new CRISPR/Cas12i3 genome editing system for wheat. This enhanced system, Opt-T5E-Cas12i3-5M, significantly improves editing efficiency in wheat, aiding crop improvement.
Area of Science:
- Plant Biotechnology
- Genome Editing
- Crop Science
Background:
- Wheat (Triticum aestivum L.) is a globally vital food crop.
- CRISPR/Cas12i3 offers advantages like smaller protein size and flexible PAM recognition.
- Previous attempts at Cas12i3-mediated editing in wheat were limited by low efficiency and the crop's hexaploid nature.
Purpose of the Study:
- To engineer a robust Cas12i3-5M genome editing system for wheat.
- To enhance editing efficiency in hexaploid wheat through system optimization.
Main Methods:
- Fusion of T5 exonuclease (T5E) to Cas12i3-5M.
- Optimization of crRNA expression strategy (Opt).
- Testing the Opt-T5E-Cas12i3-5M system on four endogenous genes in three elite Chinese wheat varieties.
Main Results:
- T5E fusion increased Cas12i3-5M editing efficiency in HEK293T cells by up to 3.87-fold.
- The optimized system (Opt-T5E-Cas12i3-5M) achieved 1.20- to 7.95-fold higher editing efficiency in wheat stable lines compared to previous versions.
- Editing efficiencies ranged from 60.71% to 90.00% across target genes in stable wheat lines.
Conclusions:
- The developed Opt-T5E-Cas12i3-5M system is a robust tool for wheat genome editing.
- This system enhances editing efficiency in polyploid wheat, overcoming previous limitations.
- The technology holds potential for accelerating wheat research and genetic improvement, and may be applicable to other polyploid crops.
