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Targeted editing of the Arabidopsis CYP98A3 gene using the CRISPR/Cas9 method
 Šamaj, Matej
Lignin is an abundant biopolymer, which fulfils several important functions including facilitation of polar water transport and mechanical strengthening of plant body. It is required for proper growth and development of plants but also for plant defence responses. Complex lignin biosynthesis involves the activity of numerous enzymes catalysing diverse chemical reactions that are stepwise generating various precursors for lignin formation. One such enzyme of lignin biosynthetic pathway, P‐COUMAROYL SHIKIMATE 3′‐HYDROXYLASE (C3′H), is encoded by the gene CYP98A3/ C3′H and convert p-coumaroyl shikimate to caffeoyl shikimate. CRISPR/Cas systems adapted from prokaryotes, where they provide adaptive immunity, have very quickly evolved to unique and powerful tool for precise gene editing. The CRISPR/Cas9 technology allows to specifically target desired sites within the genomes to subsequently perform very precise mutagenesis of selected gene(s). The purpose of this thesis was to employ the CRISPR/Cas9 method to perform targeted editing of the CYP98A3/C3′H gene in transformed Arabidopsis thaliana plants. Selected transformed plants were verified to confirm the presence of CRISPR/Cas9-mediated gene editing. The verified T2 generation putative mutant plant shows altered phenotypes of leaves, leaf rosettes, stems and inflorescences accompanied by lower deposition of lignin as revealed by histochemical staining by basic fuchsin and microscopic observations.
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1 Šamaj, Martin
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