Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential off-target effects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overexpressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infiltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a significant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efficiency of using an inducible promoter in CRISPR-Cas9 constructs.
Keywords: CRISPR-Cas9; Inducible Cas9; TYLCV; Virus resistance; rgsCaM promoter.