Disruption of Phytoene Desaturase Gene using Transient Expression of Cas9: gRNA Complex

Engineered nucleases have emerged as a powerful tool for site specific gene manipulation in plants. Based on Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR associated (CRISPR/Cas) system, engineered Cas9: gRNA complex can be used to cleave specific DNA sequences in the genome. In the present study, Nicotiana benthamiana Phytoene Desaturase (NbPDS) gene was targeted by CRISPR/Cas9 system. The plant codon optimized (pcoCas9) along with guided RNA (gRNA) was cloned in plant expression vector pGreen0029, to target NbPDS gene. The NbPDS gene was disrupted transiently by agroinfiltration of pcoCas9-gRNA complex. Visible albino spots were observed on agro-infiltrated leaves of N. benthamiana plants after 7 days of infiltration. The observed albino spots were analyzed through PCR amplification of gRNA-target, fluorescent microscopy and chlorophyll contents measurements. Our results support the notion that CRISPR/Cas9 system is a swift, robust and useful tool for targeted gene disruption, deletion and editing. (C) 2016 Friends Science Publishers