CRISPR/Cas9-mediated targeted mutagenesis in grape

• Published in: PloS one Vol. 12; no. 5; p. e0177966
• Main Authors: Nakajima, Ikuko, Ban, Yusuke, Azuma, Akifumi, Onoue, Noriyuki, Moriguchi, Takaya, Yamamoto, Toshiya, Toki, Seiichi, Endo, Masaki
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18-05-2017; Public Library of Science (PLoS)
• Subjects: Agricultural production; Agricultural research; Agriculture; Agronomy; Anthers; Aroma; Assembly; Base Sequence; Bioassays; Biology and Life Sciences; Biotechnology; Breeding; Callus; Cell lines; Cloning; Colleges & universities; Color; Computer applications; Corn; CRISPR; CRISPR-Cas Systems - genetics; Cultivars; Dehydrogenase; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA sequencing; DNA, Plant - chemistry; DNA, Plant - metabolism; Efficiency; Embryonic growth stage; Embryos; Engineering; Engineering and Technology; Filaments; Food; Fruit crops; Fruits; Functional analysis; Gene expression; Genes; Genetic aspects; Genetic Vectors - genetics; Genetic Vectors - metabolism; Genetics; Genome editing; Genomes; Grain; Grapes; Heterozygosity; Homology; Irradiation; Leaves; Livestock; Metabolism; Molecular biology; Mutagenesis; Mutation; Nucleotide sequence; Oxidoreductases - genetics; Phenotypes; Plant breeding; Plant diseases; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Proteins - genetics; Plants; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Pollen; Protoplasts; Research and Analysis Methods; Ribonucleic acid; RNA; Science; Sequence Analysis, DNA; Skin; Sorghum; Tobacco; Transcription factors; Trees; Vitis - genetics; Wine; Wines; Japan
• DOI: 10.1371/journal.pone.0177966
• EISSN: 1932-6203

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.