Efficient virus-mediated genome editing in cotton using the CRISPR/Cas9 system.

Area of Science:

• Plant biotechnology
• Molecular genetics
• Crop improvement

Background:

• Virus-mediated delivery offers advantages for gene editing due to rapid sgRNA amplification.
• Efficient gene editing requires effective delivery and expression of sgRNA in target plant cells.
• Cotton gene editing is crucial for improving this important crop.

Purpose of the Study:

• To establish and validate a virus-induced gene editing (VIGE) system in cotton using cotton leaf crumple virus (CLCrV).
• To assess the efficiency and specificity of CLCrV-mediated VIGE for targeting multiple genes.
• To explore long-distance sgRNA delivery to the shoot apical meristem (SAM) for generating mutants without tissue culture.

Main Methods:

• Development of a VIGE system in cotton overexpressing Cas9 (Cas9-OE) as the receptor.
• Utilized CLCrV to deliver sgRNAs targeting specific cotton genes (e.g., GhMAPKKK2, GhCLA1, GhPDS).
• Performed off-target effect assays and tested an FT-sgRNA strategy for SAM delivery.

Main Results:

• CLCrV-mediated VIGE successfully targeted and knocked out single genes (GhMAPKKK2, GhCLA1, GhPDS) and achieved double mutations (GhCLA1 and GhPDS).
• The system demonstrated high gene editing efficiency and specificity in cotton, with minimal off-target effects.
• The FT-sgRNA strategy enabled efficient endogenous gene editing in cotton but resulted in limited heritable mutant progeny.

Conclusions:

• The CLCrV-mediated VIGE system is an effective and efficient tool for gene editing in cotton.
• This VIGE system provides high specificity, making it valuable for precise genetic modifications.
• The CLCrV-VIGE system serves as a rapid validation tool for screening effective sgRNAs in cotton.