title: CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.
abstract: Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato.
authors.affiliation: National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Electronic address: yasuito@affrc.go.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ito
authors.affiliation: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Nishizawa-Yokoi
authors.affiliation: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Endo
authors.affiliation: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan; Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Yokohama, Kanagawa 236-0027, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Mikami
authors.affiliation: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan; Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Yokohama, Kanagawa 236-0027, Japan; Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, Kanagawa 244-0813, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Toki
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D018744:DNA, Plant / Q000235:genetics; D005638:Fruit / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D018551:Solanum lycopersicum / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010858:Pigmentation / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins
keywords: CRISPR/Cas9; Fruit ripening; Ripening inhibitor (RIN); Tomato