20.500.12556/RUL-188753 Vzpostavljanje pristopa za nevirusno urejanje genoma človeških celic γδ T s sistemom CRISPR/Cas9 Establishing an approach for nonviral genome editing of human γδ T cells with the CRISPR/Cas9 system V magistrskem delu smo razvili nevirusni pristop za urejanje genoma človeških celic γδ T z uporabo sistema CRISPR/Cas9 in ribonukleoproteinskih (RNP) kompleksov ter ovrednotili možnosti usmerjenega izbitja receptorja γδ TCR. Namen raziskave je bil vzpostaviti eksperimentalno osnovo za genski inženiring celic γδ T ter določiti učinkovitost različnih enojnih usmerjevalnih RNA (sgRNA). Z bioinformatskimi orodji smo načrtovali šest sgRNA, usmerjenih proti genom, ki kodirajo verige receptorja γδ TCR, njihovo učinkovitost pa nato eksperimentalno ovrednotili. Rezultati so pokazali, da se učinkovitost posameznih sgRNA razlikuje tudi pri usmerjenosti proti istemu genu, kar poudarja pomen izbire ustreznega tarčnega mesta in eksperimentalne validacije. Ugotovili smo, da kakovost začetnega biološkega materiala vpliva na delitev in živost celic po elektroporaciji. Analiza s pretočnim citometrom je pokazala postopno zmanjševanje izražanja γδ TCR po urejanju genoma, kar nakazuje na uspešnost izbitja tarčnih genov. Rezultati kažejo, da uporaba CRISPR/Cas9 in RNP kompleksov predstavlja izvedljiv pristop za nevirusno urejanje genoma človeških celic γδ T. Vzpostavljena metodologija lahko služi kot osnova za nadaljnje raziskave genskega inženiringa celic γδ T ter za razvoj potencialnih biomedicinskih aplikacij. In this master's thesis, we developed a nonviral approach for genome editing of human γδ T cells using the CRISPR/Cas9 system and ribonucleoprotein (RNP) complexes and evaluated the feasibility of targeted knockout of the γδ T cell receptor (γδ TCR). The aim of the study was to establish an experimental framework for the genetic engineering of γδ T cells and to evaluate the performance of different single-guide RNAs (sgRNAs). Six sgRNAs targeting genes encoding the γδ TCR chains were designed using bioinformatics tools, and their effectiveness was subsequently evaluated experimentally. The results demonstrated that the performance of individual sgRNAs varied, even when targeting the same gene, highlighting the importance of target-site selection and experimental validation. We also observed that the quality of the initial biological material could influence cell proliferation and viability following electroporation. Flow cytometry analysis revealed a gradual reduction in γδ TCR surface expression after genome editing, consistent with successful disruption of the targeted loci. The findings indicate that the use of CRISPR/Cas9 RNP complexes represents a feasible strategy for the nonviral genome editing of human γδ T cells. The established methodology may serve as a foundation for future research in γδ T cell genetic engineering and the development of potential biomedical applications. celice gama delta T preurejanje genoma CRISPR/Cas9 pretočna citometrija gamma delta T cells genome editing flow cytometry true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-27 07:16:34 2026-09-27 07:16:35 2026-09-28 04:29:56 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-25 292651 63376.pdf 63376.pdf 1 EC5930C98B16DE4F4DEEE790F06E1FC1 92fad539d6d67aeb510f57fcaf329738ff978e59e2d29bc2ef7e640754ad0c61 855d66f0-ba32-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=249079 Biotehniška fakulteta 0 0 0