CRISPR-Cas9-Mediated Editing of the CYP82E4-Nicotine N-Demethylase (nnd) Gene in Tobacco Protoplasts

Abstract

The single RNA‐guided DNA recognition CRISPR-Cas9 method is a simple and powerful tool for targeted genome engineering. Here, we report the designing and testing of efficient caulimoviral promoter-derived binary vectors for performing genome editing employing the CRISPR-Cas9-gRNA system. For such targeted mutagenesis, we created binary transformation vectors to drive the expression of Cas9 by an efficient caulimoviral promoter, ‘M24’ isolated and characterized from the Mirabilis Mosaic Virus (MMV). The 20-nucleotide CRISPR guide (g)-RNAs were designed to induce targeted mutations in the CYP82E4-nicotine N-demethylase (nnd) gene of tobacco (Nicotiana tabacum). For editing the nnd gene, we employed a pair of gRNAs followed by the protospacer adjacent motif (PAM) targeting the first exon of the nnd-ORF. We evaluated the percent “indels” using tobacco protoplast cells where mutagenesis frequencies were recorded as 45% and 30% for the two targets respectively. A mutagenesis efficiency of 37% was obtained upon the simultaneous transfection of the two gRNAs in tobacco protoplasts. Successful demonstration of our caulimoviral-based CRISPR-Cas9-gRNA system bodes well for its near-term use as a potential and facile means to performing targeted genome editing in plants.