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 To 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 wildtype 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 T-1 progeny, confirming the applicability of this mutagenesis system in tomato. (C) 2015 Elsevier Inc. All rights reserved.