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Genome editing using programmable endonucleases
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Sýkora, Michal (referee)
Programmable endonucleases are engineered proteins that recognize specific nucleotide sequences and that are capable of introducing double-strand breaks within these sequences. Zinc-finger nucleases have been used extensively as a tool in genome editing, the practice of introducing changes into genomes of cell lines or whole organisms as a way to study gene function. Recently, new types of programmable endonucleases have emerged in the form of transcription activator like effector (TALE) nucleases and the CRISPR/Cas system. The types differ in respect to their mechanism of function, accessibility, selectivity, frequency of off-target cleavage and cytotoxic effects. Here, we compare zinc-finger nucleases, TALENs and the CRISPR/Cas system and explore their current and possible future applications in a broad spectrum of research ranging from developing genetically modified organisms to gene therapy. Powered by TCPDF (www.tcpdf.org)
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The role of tissue specific isoforms of subunit 4 in assembly and function of cytochrome c oxidase
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Stibůrek, Lukáš (referee)
Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...
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Nanoparticle-Mediated Delivery System for Gene Therapy
Dvořáková, Nikola ; Ellederová, Zdeňka (advisor) ; Šálek, Petr (referee)
Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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Transdifferentiation of somatic cells into hepatocytes and clinical relevant edition of the Tight junction protein 2 gene
Fryntová, Lucie ; Janečková, Lucie (advisor) ; Krylov, Vladimír (referee)
Transdifferentiation induces chromatin reconstructions and epigenetic changes that affect gene expression spectum and cause cell remodeling in general. Direct conversion of mature somatic cell line into another mature cell type occures during the transdifferentiation thereby differences betweeen individual germ layers are eliminated. The aim of the master thesis is transdifferentation of mesenchymal cells - mouse embryonic fibroblast into endodermal cells - hepatocytes in vitro, using combination of transcripion factors Hnf4α and Foxa1. Detection of fibroblasts transformation has been initiated immediately after retroviral transduction and final generation of induced hepatocyte culture was confirmed by morphological and function analysis. The population of mouse induced hepatocytes served as a possible model for human liver disease in case of a pacient whose liver proteins could not be detected immunohistochemically. Genome editing of induced hepatocytes was realized by CRISPR/Cas9 technology which is based on cooperation of guideRNA and Cas9 nuclease followed in addition to generation of DNA-specific double strand breaks. These specific breaks in the Tight junction protein 2 gene were repaired via homologous recombination that induced a missense mutation with amino acid changes in the target...
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Tissue-specific knockout of starch synthesis in columella cells of Arabidopsis thaliana and gravitropic response
Bogdan, Michal ; Fendrych, Matyáš (advisor) ; Retzer, Katarzyna (referee)
Since the studies of plant gravitropism by Charles Darwin, the identity of specific sensors of gravity in plants has been uncertain. To this date, statoliths - starch granules in the root tips - are considered to play a key role in gravity sensing. The role of statoliths as organelles that mediate the gravity sensing ability of plant roots is based on research that uses plants which have severely impaired ability to synthesize starch in general or have their cells that contain statoliths removed or damaged. This represents methodical imperfections that give rise to alternative explanations, like disturbed auxin flow due to heavy damage to the root tip or unknown involvement of starch from other parts of the plant in gravity perception. Thanks to advances in the field of CRISPR/Cas9 technology, we are now able to produce tissue-specific mutants that might help with clarification of whether starch granules in the root tip are involved in sensing gravity and if so, how significant is this involvement. This diploma thesis aimed to answer these questions by adapting the tissue-specific CRISPR/Cas9 system and using it for the creation of mutants that are starchless specifically in the columella cells. Using this approach, we generated one tissue non-specific mutant line and three tissue-specific mutant...
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Dynamics of de novo DNA methylation and its impact on transgene expression and CRISPR/Cas9 mutagenesis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Fajkus, Jiří (referee)
Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...
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Checkpoint blockade in cancer immunotherapy
Vacková, Julie ; Šmahel, Michal (advisor) ; Černý, Jan (referee) ; Říhová, Blanka (referee)
The immune checkpoint blockade is a novel approach of cancer therapy, which markedly enhanced treatment efficacy of several cancer types. However, the frequency of cancer patients non-responding to this treatment is high. Establishment of predictive markers to distinguish patients suitable for the immune checkpoint blockade would enhance the number of patients receiving benefit from the therapy. This dissertation thesis focuses on the enhancement of efficacy of immune checkpoint inhibitors (ICIs) and predictive markers in experimental models of mouse tumours induced by TC-1 and TC-1/A9 cell lines and its clones with deactivation of interferon (IFN)-γ signalling (TC-1/dIfngr1 and TC-1/A9/dIfngr1), or CD80 molecule (TC-1/dCD80-1). IFN-γ is presumed to be the main inducer of programmed death ligand 1 (PD-L1) and a major histocompatibility complex I (MHC-I). Moreover, PD-L1 expression may predict sensitivity to PD-1/PD-L1 blockade. Non-functional IFN-γ signalling or downregulated MHC-I expression has been associated with resistance to ICIs in some patients. We found that IFNs type I (IFN-α and IFN-β) induced the expression of PD-L1 and MHC-I on TC-1/A9/dIfngr1 tumour cells with reversible downregulation of both molecules. We also showed that deactivation of IFN-γ signalling in TC-1/A9 cells was not a...
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Generation and analysis of mutant mouse model to study roles of KLKs in cutaneous inflammation
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Drbal, Karel (referee)
Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases of undisputable importance for a variety of functions, whose dysregulation has been linked to several pathological phenotypes. Among those pathologies, the Netherton syndrome stands out, since it is one of the very few that has its mechanism directly linked to KLK proteases as the main culprit of the disease, namely KLK5, KLK7 and to a lesser degree, KLK14. In this case, a mutation in the SPINK5 gene leads to uncontrolled hyperactivity of those proteases, which results in epidermal barrier breach due to excessive epidermal desquamation and severe inflammation of the skin. Inflammation mechanisms of NS are still relatively poorly understood, with important roles being attributed to the activities of KLKs in the processing of immune system molecules and also to the dysregulation of the cutaneous microbiome. TNFα signalling plays a key role in the homeostasis and immune response in the skin. Chronic skin infections may lead to deleterious effects with strong participation of TNFα signalling. To address the degree of its effects on the pathogenesis of NS, we have created a mouse model where the TNFR1 is disrupted by knockout of the Tnfr1 gene on the background of a previously established mouse model of the Netherton syndrome. We...
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