Efficient CRISPR-Cas9-Mediated Genome Editing in Tomato

To understand gene function and create novel traits in plants, use of the powerful clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system has become commonplace around the world. This system has been adopted in several plant species. In this protocol, we describe, in a step-wise manner, guidelines for the selection of a suitable vector and target sites, design of sgRNAs, including analysis of off-target activity, construct design, and its application in cultivated tomato (Ailsa Craig). Apart from presenting a method for the CRISPR-Cas-mediated genome editing, we also provide a detailed protocol for highly efficient plant transformation and regeneration in tomato to generate stable lines along with variant analysis of putative mutant lines followed by phenotypic analysis for the trait-of-interest. From the design of target site, generation of genome-edited tomato plants can be realized in 16–20 weeks.