CRISPR/Cas9 is gaining importance as a gene engineering tool for targeted gene modifications of plant genomes. Use of PEG-stimulated sgRNA and Cas9 protein direct uptake into protoplasts is one of the methods that enable transgene-free targeted mutagenesis. The aim of our work was to deliver ribonucleoprotein complexes into rapeseed (Brassica napus L.) protoplasts using PEG and in this manner induce target specific mutations. For this purpose we optimized preparation of plant material, composition of enzyme solution for degradation of cell walls and the procedure of protoplasts isolation. We found that etiolated hypocotyls are the most suitable plant material and that cellulases Onozuka R-10 and Onozuka RS are the most effective for degradation of cell walls. The most appropriate isolation protocol was chosen based on protoplasts viability and concentration and also on successful insertion of expression plasmid containing reporter gene ZsGreen. For targeted mutagenesis we used four different sgRNAs with targets in pds gene, in complex with Cas9 enzyme. After 48 hours of incubation we isolated DNA from protoplasts. To identify possible target mutations we used T7 endonuclease assay, RFLP analysis and Sanger sequencing. The results of enzyme tests indicated successful induction of targeted mutations, but could not be reliably verified with Sanger sequencing. In order to obtain better results further optimization of protoplast isolation and/or transformation procces is needed and also the use of more sensitive methods for identification of targeted mutations.
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