CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation

The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is a powerful tool for editing plant genomes. Efficient genome editing of grape (Vitis vinifera) suspension cells using the type II CRISPR/Cas9 system has been demonstrated; however, it has not been established whether this system can be applied to get biallelic mutations in the first generation of grape. In this current study, we designed four guide RNAs for the VvWRKY52 transcription factor gene for using with the CRISPR/Cas9 system, and obtained transgenic plants via Agrobacterium-mediated transformation, using somatic embryos of the Thompson Seedless cultivar. Analysis of the first-generation transgenic plants verified 22 mutant plants of the 72 T-DNA-inserted plants. Of these, 15 lines carried biallelic mutations and seven were heterozygous. A range of RNA-guided editing events, including large deletions, were found in the mutant plants, while smaller deletions comprised the majority of the detected mutations. Sequencing of potential off-target sites for all four targets revealed no off-target events. In addition, knockout of VvWRKY52 in grape increased the resistance to Botrytis cinerea. We conclude that the CRISPR/Cas9 system allows precise genome editing in the first generation of grape and represents a useful tool for gene functional analysis and grape molecular breeding.