Genome editing in PDS genes of tomatoes by non-selection method and of Nicotiana benthamiana by one single guide RNA to edit two orthologs

The CRISPR/Cas9 system is widely used for targeted mutagenesis in many organisms including plants. For application of this system, tissue culture methods need to be established. In this study, detailed methods for introduction of mutations in tomato and Nicotiana benthamiana plants using the CRISPR/Cas9 system are described. The methods include tissue culture protocols for tomato and N. benthamiana. We also demonstrate the methodology to generate Cas9-free genome edited tomato plants and use of one single guide RNA (sgRNA) to edit two orthologs in N. benthamiana. The examples of editing the PHYTOENE DESATURASE (PDS) genes in these plants are also provided. The Cas9-free tomato line was obtained when tomato plants were cultured on a non-selective medium after transformation with the CRISPR/Cas9 system. Two orthologs of PDS in N. benthamiana were mutated using a sgRNA, because these orthologs contain the same nucleotide sequences with PAM motif. These mutations were inherited to the next generation. The mutations in the PDS genes resulted in an albino phenotype in tomato and N. benthamiana plants. These results demonstrate that the non-selective method is one of the ways to obtain Cas9-free genome editing in tomato plants and that the two orthologs can be edited by one sgRNA in N. benthamiana.