Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters.

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Agricultural biotechnology

Background:

• CRISPR/Cas9 efficacy in grapevine is known, but optimization and multiplex editing remain underexplored.
• Efficient gene expression is crucial for successful genome editing.

Purpose of the Study:

• To optimize the CRISPR/Cas9 system for grapevine genome editing.
• To develop and apply multiplex genome editing strategies in Vitis vinifera.
• To investigate the role of TMT1 and TMT2 genes in grape sugar accumulation.

Main Methods:

• Identification and characterization of four VvU3/U6 and two UBQ promoters in grapevine.
• Construction of optimized CRISPR/Cas9 vectors for enhanced sgRNA and Cas9 expression.
• Multiplex genome editing of TMT1 and TMT2 genes using conventional or PTG cassettes.
• Validation of promoter activity in tobacco (Nicotiana benthamiana) by editing the PDS gene.

Main Results:

• VvU3/U6 and UBQ2 promoters significantly enhanced editing efficiency by improving sgRNA and Cas9 expression.
• Multiplex editing of TMT1 and TMT2 achieved efficiencies exceeding 10%.
• Simultaneous editing of TMT1 and TMT2 led to reduced sugar levels in grapes.
• Demonstrated cross-species applicability of promoters in tobacco genome editing.

Conclusions:

• Optimized CRISPR/Cas9 system with identified promoters improves gene editing efficiency in grapevines.
• Multiplex editing of TMT1 and TMT2 provides insights into grape sugar accumulation mechanisms.
• This study establishes the first multiplex genome editing of grape TMT family genes, expanding tools for grapevine research and breeding.