Efficient Multiplex Genome Editing Induces Precise, and Self-Ligated Type Mutations in Tomato Plants.

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Biotechnology

Background:

• CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) enables multiplex gene editing in plants.
• Various expression systems for multiple guide RNAs (gRNAs) exist for plant gene modification.

Purpose of the Study:

• To evaluate mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors.
• To compare different Cas9 expression promoters for their effectiveness in multiplex genome editing.

Main Methods:

• Transgenic tomato calli were generated using multiplex CRISPR/Cas9 vectors with various Cas9 promoters and multiple gRNA combinations.
• Mutation patterns and efficiencies were analyzed in the tomato genome.
• Sequence analysis was performed to characterize mutation types and frequencies.

Main Results:

• Cas9 expression promoter choice significantly influenced mutation patterns in the tomato genome.
• The tomato ELONGATION FACTOR-1α (SlEF1α) promoter demonstrated high efficiency for various mutation types.
• Multiplex editing with the SlEF1α promoter resulted in deletions via self-ligation at target sites with low mosaic mutations.

Conclusions:

• Optimizing the Cas9 expression promoter is crucial for improving multiplex genome editing efficiency in plants.
• The SlEF1α promoter is effective for precise disruption of functional domains in the tomato genome using CRISPR/Cas9.