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CRISPR/Cas genome editing in human disease models and the translation into therapies
Faltínová, Pavlína ; Krupková, Michaela (advisor) ; Šedová, Lucie (referee)
Rare diseases, as their name indicates, individually affect only a low number of people around the world. Due to their low prevalence, finding appropriate therapy is very difficult. Insufficient understanding of the molecular causalities and mechanisms accompanying these disorders and the inability to conduct clinical studies to the usual extent due to the low occurrence of rare diseases belong to the main problems hindering the development of proper treatment. The creation of mouse models is a promising way to solve these difficulties since mice have numerous qualities necessary for modelling human diseases. CRISPR/Cas9 enables scientists to make precise changes in the genome by employing Cas9 nuclease which creates double-strand breaks in the DNA after the specifically designed guide RNA leads it to the site of interest. If the sequence of the chosen site is known, the possible edits can be installed almost anywhere in the genome and, moreover, their repertoire is practically endless. The use of the CRISPR/Cas9 technology proved to be perfect for creating mouse models of rare diseases as most of these disorders are caused by genetic mutations that this method is fully capable of mimicking. This thesis focuses on strategies used in creating such mouse models with the CRISPR/Cas9 system and...
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Knockout isoforem metalothioneinu pomocí metody CRISPR-Cas9 u adherentních tkáňových kultur
Duda, Jakub
It is not too long ago, that genome modifications were very complex, or, in some cases, almost impossible to achieve, but with the discovery and description of the CRISPR system in prokaryotes, a lot of breakthroughs came to the human knowledge, and thanks to some of these breakthroughs the CRISPR system has been modified to be used as a genome editing tool in eukaryotic organisms. Genetic modifications could in theory be the key to cancer therapy in modern pharmacy, and this thesis focuses on Metalmetallothionein (MT) proteins, which are commonly found in organisms and are beneficial to the organism (as inhibotors of oxidative stress, or as antioxidants binding heavy metals, for example), but can also be dangerous. Because, according to the isoform, and also the type of tissue, MTs can cause the development of cancer tissue and tumour growth, sometimes because of their presence, in other cases because of their absence. This thesis was focusing on the possible knock-out of all isoforms of MT at once, using CRISPR/Cas9 method, and thereby infucencing one of the very important factors in carcinogenesis. The result of this thesis is the achievement of knock-out of the MT3 isoform.
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Různé metodické přístupy za účelem snížení exprese podjednotek komplexu I u hmyzích forem \kur{Trypanosoma brucei}
HEROUTOVÁ, Barbora
Complex I (NADH:ubiquinone dehydrogenase) is the largest protein complex of the mitochondrial electron transport chain, but its presence and activity are not essential for the growth of two life cycle stages of Trypanosoma brucei. Here, we implemented various genetic methods to generate T. brucei cell lines that will lack or will have decreased levels of this complex. The methodological approaches included i) RNA interference of NDUFA6, a subunit predicted to be essential for the structural integrity, and of NUBM, a subunit essential for the complex I activity; ii) generation of double knock-out of NDUFA6 using CRISPR/Cas9; iii) generation of double knock-out of NUBM using homology replacement. These generated tools will help us to elucidate if this highly conserved complex is essential for other life cycle stages of this medically important parasite
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Development of new molecular tools for altering gene expression in Giardia intestinalis .
Horáčková, Vendula ; Doležal, Pavel (advisor) ; Horváthová, Lenka (referee)
Giardia intestinalis is a widespread intestinal parasite that causes diarrhea in human and other vertebrate hosts. Although, fully sequenced genome of G. intestinalis has been published, very little is known about the regulation of gene expression. Together with the tetraploid genome, this complicates the use of many common reverse genetics methods. The aim of this thesis was to develop new molecular tools that can be used to alter gene expression in G. intestinalis. For the purposes of this work, new vectors for tetracycline-inducible gene expression including T7 promoter and endogenous oct promoter were designed. Furthermore, cwp1 gene knock-out was created using CRISPR-Cas9 technology. In order to modify mechanisms of double strand break repair, expression of two key components of bacterial NHEJ pathway - LigD and Ku - was introduced into cells of G. intestinalis.
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Genes participating in response to Leishmania major revealed by targeted mutation.
Ezrová, Zuzana ; Lipoldová, Marie (advisor) ; Krulová, Magdaléna (referee)
Leishmania major is an intracellular parasite which often successfully multiplies and disseminates in a body of the host thanks to strategies that help it to escape the components of immune system of the host organism. Thus, the parasites evoke an impairment of regulatory pathways that in physiological conditions lead to an expression of genes involved in a response to L. major and its efficient elimination. Gene targeted deletion, also called gene knock-out, can result in phenotypic alteration and associated enhanced susceptibility or resistance of the host. Although such detected genes do not have to signify their variability in population and hence they may not be responsible for the worsened outcome of leishmaniasis of some people necessarily, studies in which they are analysed and general knowledge being also a subject of this thesis help us together with techniques of forward genetics to reveal the biochemical pathways during the infection and their elements that influence the outcome of the disease and might be useful for researches of new medicine drugs or gene therapy.
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