Site-directed mutagenesis offers an efficient way of studying gene function and fundamental biological processes. A way to edit multiallelic organisms is CRISPR Cas9, one of the newest methods for genome editing. In our research we have optimised CRISPR Cas9 for gene knockouts in potato. We chose the StRbohD gene that encodes an NADPH oxidase which plays a role in defence of potato against the potato virus Y. Silencing of this gene by short hairpin RNA causes loss of resistance of potato cultivar Rywal to PVY. Single guide RNA was designed to target a region, crucial for the function of StRbohD gene. We introduced sgRNA into the plasmid pRGEB31, which also contained the sequence of Cas9 protein. We performed agrobacteria-mediated stable transformation of potato cultivar Rywal using the prepared construct. We collected 70 lines of transformed shoots. We randomly chose 36 lines and inoculate them with PVY N605-GFP to study the effect of StRbohD knock-out on virus resistance. Lesions appeared on upper non-inoculated leaves in 5 transgenic lines and we confirmed the presence of viral RNA with qPCR. Furthermore, we studied progress of lesion appearance in time in two transgenic lines. One of them outnumbered the other lines by the number of lesions which also had different phenotype in comparison to the lesions that were developed in control plants. Sanger sequencing didn't reveal any mutations in the targeted region, therefore sequencing of wider area of the gene should be performed.
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