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.
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