Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 genome editing is widely used but challenged in triploid species like banana due to low efficiency.
• Developing efficient genome editing in banana is crucial for crop improvement and functional genomics.

Purpose of the Study:

• To enhance CRISPR/Cas9 genome editing efficiency in banana.
• To establish a method for validating genome editing efficiency in banana protoplasts.
• To lay the groundwork for DNA-free genome editing in banana.

Main Methods:

• Utilized an endogenous U6 promoter (MaU6c) and banana codon-optimized Cas9.
• Developed a transient expression system in banana protoplasts for efficiency validation.
• Compared the activity of MaU6c with OsU6a promoters.

Main Results:

• The MaU6c promoter showed approximately four times higher activity than the OsU6a promoter in banana protoplasts.
• The optimized CRISPR/Cas9 system, using MaU6c and banana codon-optimized Cas9, resulted in a fourfold increase in mutation efficiency.
• A reliable method for validating genome editing efficiency in banana protoplasts was established.

Conclusions:

• The optimized CRISPR/Cas9 system is effective for targeted gene mutation in banana.
• These advancements will improve functional genomics studies and germplasm improvement in banana.
• The study provides a foundation for developing DNA-free genome editing technology in banana.