Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• CRISPR-based directed evolution is a key tool for improving plant agronomic traits.
• Current methods using single guide RNAs limit gene diversification.

Purpose of the Study:

• To develop a novel CRISPR-based system for maximizing gene diversification.
• To enhance the efficiency of base editing for targeted trait improvement in plants.

Main Methods:

• Developed a multiplexed orthogonal base editor (MoBE) system.
• Implemented a randomly multiplexed sgRNA assembly strategy to increase target combinations.
• Applied MoBE and sgRNA libraries for directed evolution of the OsACC gene in rice.

Main Results:

• MoBE demonstrated efficient orthogonal base editing (ABE, CBE, A&CBE) on multiple targets.
• The sgRNA strategy generated over 27,000 target-scaffold combinations for gene editing.
• Directed evolution using MoBE resulted in rice with enhanced herbicide resistance due to specific mutations.

Conclusions:

• The MoBE system and random sgRNA assembly strategy significantly expand gene diversification capabilities.
• These tools facilitate in situ directed evolution for rapid trait improvement in crops like rice.
• This approach holds promise for accelerating the development of agriculturally important plant varieties.