Editing of Nitrate Reductase Gene Targeted By CRISPR/Cas9 in Petunia Protoplast System

Target mutagenesis of genes associated with preferred traits has been advancing continuously and precise technique applicable to genome modification of plant has been introduced recently. A CRISPR/Cas9 system has been recently announced as a powerful molecular breeding tool for site directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis of nitrate reductase gene using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia protoplast system. After protoplasts isolation, washing and enzyme digestion processes, the resuspended protoplasts were transfected with Cas9 protein and sgRNA. Genomic DNA was extracted from transfected protoplasts for T7E1 assay and targeted deep sequencing of the target locus. The RGEN RNPs induced site-specific mutations with maximum 21% at four different sites in the PhNR gene locus from the T7E1 assay. Targeted deep DNA sequencing revealed a mutation rate up to 17.8% with an average mutation rate of 11.5% at the same NR gene of the analyzed protoplast transfectants. Results also showed that the CRISPR/Cas9 system induced insertion or deletion in four out of six specific sites of the NR gene in the genome of Petunia, demonstrating that direct delivery of RGEN RNPs into protoplast cells of Petuniacan be used as an efficient tool for site-directed mutagenesis of gene of interest in the plant.