Optimisation of CRISPR Cas9 technology for gene editing in potato

Tarčno izbijanje ali mutageneza nam omogoča študij funkcije posameznega gena. Namen naše raziskave je bil optimizirati metodo za izbijanje genov CRISPR Cas9 pri krompirju. Za tarčni gen smo si izbrali gen StRbohD, ki nosi zapis za oksidazo NADPH, ki je vključena v odziv krompirja na krompirjev virus Y. Utišanje tega gen s tehnologijo kratkih lasničnih RNA vodi v slabšo odpornost rastlin Rywal na PVY. Virus se po okužbi teh rastlin lahko razširi v zgornje neokužene liste, pri čemer pride do nastanka nekrotičnih lezij. Načrtovano vodilno RNA, ki je homologna delu zaporedja gena StRbohD, smo vnesli v plazmid pRGEB31, ki med drugim vsebuje tudi zapis za encim Cas9. S konstruktom smo transformirali krompir sorte Rywal. Iz transformiranih poganjkov smo vzgojili 70 linij. 36 naključno izbranih linij smo okužili z virusom PVY N605-GFP in spremljali pojav simptomov. Pet linij je razvilo lezije v zgornjih neinokuliranih listih. Prisotnost virusne RNA smo potrdili tudi s qPCR. Pri dveh linijah smo spremljali število lezij na okuženih listih v daljšem časovnem obdobju. Ena od linij je izstopala po visokem številu lezij na okuženih listih, ki je primerljivo oziroma večje od števila lezij na okuženih listih krompirja genotipa NahG-Rywal, občutljivega na okužbo s PVY. S sekvenciranjem tarčnega zaporedja gena StRbohD testiranih linij nismo našli sprememb, zato bo potrebno spremembe poiskati v širšem območju tarčnega gena.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\u27t reveal any mutations in the targeted region, therefore sequencing of wider area of the gene should be performed