Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant

Area of Science:

• Plant Genomics
• Molecular Biology
• Biotechnology

Background:

• Upland cotton (Gossypium hirsutum) is an allotetraploid with complex subgenomes (At and Dt).
• Gene functional investigation in cotton is challenging due to gene redundancy from multiple homologous copies.
• Effective functional genomics techniques are crucial for cotton research and breeding.

Purpose of the Study:

• To evaluate two novel genome editing vectors against a previously developed CRISPR/Cas9 system in cotton.
• To compare the editing efficiency and specificity of these vectors for functional genomics in upland cotton.

Main Methods:

• Developed two novel genome editing vectors: pBeYDV-Cas9-KO and pRGEB32-35S.
• Compared these vectors with the established pRGEB32-GhU6.7 CRISPR/Cas9 system.
• Assessed editing efficiency of the GhCLA1 gene in both At and Dt subgenomes of transgenic cotton calli/plants.

Main Results:

• The pBeYDV-Cas9-KO vector demonstrated the highest simultaneous editing efficiency (73.3%) for GhCLA1 in both subgenomes.
• Individual subgenome editing efficiencies for pBeYDV-Cas9-KO reached 85.7% (At) and 97.2% (Dt).
• pBeYDV-Cas9-KO editing primarily involved fragment deletions (84.0%) within a 1-10 bp range, with no detected off-target effects.

Conclusions:

• The pBeYDV-Cas9-KO system exhibits superior editing efficiency and specificity compared to traditional CRISPR/Cas9.
• This novel vector provides a powerful and precise tool for advancing cotton functional genomics and molecular breeding.
• The system's effectiveness in simultaneous editing of homologous genes in allotetraploid cotton is a significant advancement.