Efficiency Optimization of CRISPR/Cas9-Mediated Targeted Mutagenesis in Grape

Area of Science:

• Agricultural Science
• Molecular Biology
• Genetics

Background:

• The Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas system offers efficient genome editing.
• CRISPR/Cas9 has been applied to grape (Vitis vinifera), but its efficiency requires optimization.

Purpose of the Study:

• To optimize CRISPR/Cas9 genome editing efficiency in Vitis vinifera.
• To investigate the impact of sgRNA GC content, cell variety, and SpCas9 expression on editing efficiency.

Main Methods:

• Designed four sgRNAs targeting the Vitis vinifera phytoene desaturase gene with varying GC content.
• Utilized suspension cells from 'Chardonnay' and '41B' grape varieties for transformation.
• Assessed editing efficiency using T7EI and PCR/RE assays.

Main Results:

• Editing efficiency increased proportionally with sgRNA GC content, peaking at 65% GC.
• The '41B' variety exhibited higher gene editing efficiency than 'Chardonnay'.
• SpCas9 expression levels influenced editing efficiency less than sgRNA GC content.

Conclusions:

• sgRNA GC content and cell variety are key factors for optimizing CRISPR/Cas9 editing in grapes.
• A 65% GC content for sgRNA and the '41B' variety are recommended for enhanced grape genome editing.
• Findings provide a basis for improving CRISPR/Cas9 applications in Vitis vinifera.