CRISPR/Cas9-Targeted Gene Modification in Solanaceae Crops

A CRISPR/Cas9 system has been established in Solanaceae crops petunia (Petunia hybrida cv. midnight) and tomato (Solanum lycopersicum cv. ) in our laboratory using a direct delivery of RGEN ribonucleoproteins (RNPs)–purified Cas9 protein along with target specific in vitro transcribed sgRNA. Here, we further described Cas9/sgRNA technology in tomato protoplast system to establish a site-directed mutagenesis for eukaryotic translation initiation factor 4E (eIF4E) gene which leads to virus resistance in tomato cultivars. Also a gene flavanone 3-hydroxylase (F3H) involving in anthocyanin biosynthesis was targeted in order to modify the flower color using the same RNP system. Petunia and tomato protoplasts were isolated from in vitro grown plants after germination in MS medium. After evaluating the isolated protoplasts under microscope, the highest transformation condition was optimized by using 40% of PEG-4000 with 24 hr incubation time, in which approximately 40% of the transfected cells showed the GFP expression. Following direct delivery of RNP complex (Cas9 protein + sgRNA) to petunia and tomato protoplast at 24 hour incubation, RGEN mediated various insertions or deletions (‘‘indel’’) in exons of F3H and eIF4E gene loci were validated by T7E1 assay and frequency of Indel rate was further estimated by target deep sequencing. Here we demonstrate the development of an efficient site-directed mutagenesis in petunia F3H and tomato eIF4E genes using direct delivery of engineered RNPs.