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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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triggers of asexual reproduction: on the crosslink between hybridization, asexuality, polyploidy and speciation on example of Cobitidae loaches
Marta, Anatolie ; Janko, Karel (advisor) ; Arai, Katsutoshi (referee) ; Trachtulec, Zdeněk (referee)
(in English) Sexual reproduction is considered a nearly universal feature of all eukaryotic organisms and has been hypothesized to be their ancestral state. Sexual reproduction is mainly represented by meiotic division, recombination, production of haploid gametes and fertilization. Although molecular and cytological mechanisms underlying meiosis are highly conservative they may be disrupted in numerous ways leading to the emergence of so-called asexual lineages. The proximate origins of asexuality may differ for particular taxa. In vertebrates, asexuality frequently is triggered by interspecific hybridization. Nowadays "classical" theories predict that asexuals should not be able to persist on a long-term evolutionary scale. However, the hybrid lineages have to overcome short-term disadvantages, such as postzygotic barriers ranging from complete hybrid sterility to altered meiosis resulting in asexual reproduction and even ploidy elevation. Despite that hybrid sterility is one of the most common outcomes of interspecific hybridization, however various lineages found their ways to alleviate these problems and produce viable clonal gametes. The knowledge about proximate mechanisms of unreduced gamete formation in asexual lineages is very limited as many studies were restricted due to methodological...
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CRISPR/Cas9 gene editing in Drosophila melanogaster
KALTENBÖCK, Konstantin
The aim of this thesis was to predict functional sites in the timeless gene of Drosophila melanogaster, to design fitting gRNAs for the identified sites to create transformation vectors for fly transformations and to induce CRISPR/Cas9 mediated target mutations by crossing gRNA expressing flies with Cas9 expressing flies.
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Nanoparticles for gene editing
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Beranová, Jana (referee)
Early DNA-based therapies were tested for therapeutic applications, but they sooner or later revealed multiple hurdles and risks preventing their use in further clinical trials. Recently, they have been replaced by rapidly evolving gene editing using programmed nucleases capable of precise genome modifications by cleaving specific DNA sequences. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 system are currently under investigation as potential therapeutics. However, their off-target effects must be controlled. Targeted delivery of nucleases in a form of mRNA seems as the most promising method. Various types of nanoparticles enable mRNA transfer and could be used to facilitate the nuclease application. Some of these nanoparticles together with characterization of the programmed nucleases are described in this thesis.
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Porcine models for Huntington disease
Růna Vochozková, Petra ; Motlík, Jan (advisor) ; Bohačiaková, Dáša (referee) ; Fulková, Helena (referee)
The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...
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Porcine models for Huntington disease
Růna Vochozková, Petra ; Motlík, Jan (advisor) ; Bohačiaková, Dáša (referee) ; Fulková, Helena (referee)
The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...
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Nanoparticles for gene editing
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Beranová, Jana (referee)
Early DNA-based therapies were tested for therapeutic applications, but they sooner or later revealed multiple hurdles and risks preventing their use in further clinical trials. Recently, they have been replaced by rapidly evolving gene editing using programmed nucleases capable of precise genome modifications by cleaving specific DNA sequences. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 system are currently under investigation as potential therapeutics. However, their off-target effects must be controlled. Targeted delivery of nucleases in a form of mRNA seems as the most promising method. Various types of nanoparticles enable mRNA transfer and could be used to facilitate the nuclease application. Some of these nanoparticles together with characterization of the programmed nucleases are described in this thesis.
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TAL Effectors: Tools for DNA Targeting
Jankele, Radek ; Svoboda, Petr (advisor) ; Černý, Jan (referee)
Two decades of research on interactions between Xanthomonas phytopathogenic bacteria and their hosts resulted in discovery of a novel Transcription Activator-Like Effector (TALE) protein family, which confers bacterial virulence in plants. TALEs bind selectively to plant promoters and activate expression of cognate genes enabling bacterial reproduction and dissemination. TALEs mediate recognition of specific promoter boxes in a simple and predictable manner. The TALE central repeat domain contains tandem repeats, which specifically contact single consecutive nucleotides in the target sequence via polymorphic amino acid residues. Repeats stack together in an unique right-handed superhelical assembly, which wraps around the DNA duplex. Validated TALE-DNA binding code shows, that two polymorphic amino acids NI, HD, NH, NG and NN in each repeat mediate recognition of A, C, G, T and A/G, respectively. The order of repeats determines recognized sequence in DNA sense strand. Custom TALE DNA-binding domains with desired specificities can be created within one week at low cost. Such designed domains fused to nuclease or activation domains are useful in research, biotechnology and gene-therapy for targeted gene editing and gene regulation. Notably, gene editing with custom-designed TALE nucleases (TALENs) allows for...
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