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

Abstract

Clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system is an efficient targeted genome editing method. Although CRISPR/Cas9-mediated mutagenesis has been applied successfully in grape, few studies have examined the technique's efficiency. To optimize CRISPR/Cas9 editing efficiency in <i>Vitis vinifera</i>, we surveyed three key parameters: GC content of single guide RNA (sgRNA), variety of transformant cells used, and <i>SpCas9</i> expression levels in transgenic cell mass. Four sgRNAs with differing GC content were designed to target exon sites of the <i>V. vinifera</i> phytoene desaturase gene. Suspension cells of 'Chardonnay' and '41B' varieties were used as the transgenic cell mass. Both T7EI and PCR/RE assays showed that CRISPR/Cas9 editing efficiency increases proportionally with sgRNA GC content with 65% GC content yielding highest editing efficiency in both varieties. Additionally, gene editing was more efficient in '41B' than in 'Chardonnay.' CRISPR/Cas9 systems with different editing efficiency showed different <i>SpCas9</i> expression level, but compared with GC content of sgRNA, <i>SpCas9</i> expression level has less influence on editing efficiency. Taken together, these results help optimize of CRISPR/Cas9 performance in grape.