Development of Potyvirus Resistance in Non-transgenic Tomato Plants Using CRISPR/Cas9 Technology

Tomato (Solanum lycopersicum L.) belongs to the Solanaceae, which is economically an important vegetable crop and highly vulnerable to viruses in the potyvirus family. The eukaryotic translation initiation factor eIF4E family have been found as major determinants of plant susceptibility to potyviruses. However, the identification and functional characterization of these eIF4E genes in important crop is often limited. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system has been emerged as a powerful molecular breeding tool for site directed mutagenesis in higher plants. To investigate the further role of eIF4E in virus resistance of tomato we set up a CRISPR/Cas9 system in four different cultivars (Summer King, Sweet King, Minipop and Micro Tom) to mutate normal function of eIF4E using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into isolated protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting eIF4E, mutagenesis at the targeted loci of tomato cultivars of protoplast transfectants were confirmed by cloning of eIF4E loci and targeted deep sequencing. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the cloned nucleotide sequences of eIF4Es of protoplast transfectants, but not in putative off-target sites. Targeted deep DNA sequencing analysis revealed mutation rates produced collectively by the three eIF4E-RGEN target sites were ranged from 2% to 6.8% with an average of 3.8 ± 1.01% in DNA fragments of analyzed protoplast transfectants of Minipop cultivar. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of tomato can be exploited as an efficient tool for generating potyvirus resistance genes in important crops without the use of transgenic approaches.