Journal of Plant Biochemistry & Physiology
Open Access
ISSN: 2329-9029
+44 1478 350008
ISSN: 2329-9029
+44 1478 350008
Nrisingha Dey
Institute of Life Sciences, Chandrasekharpur,
Bhubaneswar Odisha
India
Research Article
CRISPR-Cas9-Mediated Editing of the CYP82E4-Nicotine N-Demethylase (nnd) Gene in Tobacco Protoplasts
Author(s): Ankita Shrestha, Ahamed Khan and Nrisingha DeyAnkita Shrestha, Ahamed Khan and Nrisingha Dey
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 &ld.. View More»
DOI:
10.4172/2329-9029.1000230