Removal of a 10-kb Transposon from of Cv. Chardonnay

Many white grape cultivars have a nonfunctional gene due to the presence of a 10-kb transposon in its promoter. In this study, we successfully demonstrated removal of the 10-kb transposon and functional restoration of a allele in cv. Chardonnay through transgenic expression of Cas9 and two gRNAs simultaneously targeting two junction sequences between LTRs and . We generated 67 and 24 Cas9-positive vines via -mediated and biolistic bombardment transformation, respectively. While the editing efficiencies were as high as 17% for the 5' target site and 65% for the 3' target site, simultaneous editing of both 5' and 3' target sites resulting in the removal of transposon from the promoter was 0.5% or less in most transgenic calli, suggesting that these calli had very limited numbers of cells with the removed. Nevertheless, two bombardment-transformed vines, which shared the same unique editing features and were likely derived from a singly edited event, were found to have the successfully edited out from one of their two alleles. The edited allele was functionally restored based on the detection of its expression and a positive coloring assay result in leaves. Precise removal of more than a 10-kb DNA fragment from a gene locus in grape broadens the possibilities of using gene editing technologies to modify various trait genes in grapes and other plants.