Immunotherapy presents one of the promising approaches in modern cancer treatment, as it relies on redirecting and enhancing the patient’s own immune system to specifically recognize and eliminate malignant cells. A key strategy within this field is the genetic engineering of T cells, where technologies such as CRISPR/Cas enable precise and efficient reprogramming of immune cells for therapeutic use. In this master’s thesis, we developed and optimized a non-viral genome editing methodology in the Jurkat T-cell line. Using ribonucleoprotein (RNP) complexes composed of Cas9 and single guide RNA (sgRNA), we achieved efficient knockout of the target gene TRAC, which encodes a component of the T-cell receptor (TCR). Subsequently, through homology-directed repair (HDR), we introduced a sequence for the reporter protein GFP into the same genomic locus. The efficiency and stability of the modifications were monitored by flow cytometry. Our results confirm that the presented methodology enables both efficient gene knockout and targeted insertion of desired genetic constructs, representing an important step toward the development of non-viral strategies for T-cell genetic engineering in the context of cellular cancer immunotherapy.
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