20.500.12556/RUL-173932 Nevirusno urejanje genoma T-celične linije Jurkat za izbitje tarčnega gena TRAC in vnos zapisa za zeleni fluorescenčni protein z uporabo sistema CRISPR/Cas9 Non-viral genome editing in Jurkat T-cell line for targeted knockout of TRAC gene and insertion of the green fluorescent protein gene with CRISPR/Cas9 Imunoterapija predstavlja enega obetavnejših pristopov v sodobnem zdravljenju raka, saj temelji na preusmerjanju in krepitvi bolnikovega lastnega imunskega sistema za specifično prepoznavanje in uničevanje rakavih celic. Pomembno strategijo na tem področju predstavlja genetsko inženirstvo celic T, kjer tehnologije, kot je CRISPR/Cas, omogočajo natančno in učinkovito spreminjanje imunskih celic za terapevtsko uporabo. V tem magistrskem delu smo razvili in optimizirali nevirusno metodologijo za urejanje genoma v celični liniji Jurkat, ki predstavlja model za celice T. Z uporabo ribonukleoproteinskih (RNP) kompleksov, sestavljenih iz Cas9 in enojne usmerjevalne RNA (sgRNA), smo uspešno izvedli izbitje tarčnega gena TRAC, ki kodira komponento T-celičnega receptorja (TCR). Nadalje smo s posredovanjem popravljanja na osnovi homologije (HDR) v isti genomski lokus uspešno vstavili zapis za reporterski protein GFP. Učinkovitost in stabilnost genetskih sprememb smo spremljali s pretočno citometrijo. Rezultati potrjujejo, da predstavljena metodologija omogoča tako učinkovito izbitje genov kot tudi tarčno integracijo želenega konstrukta, kar predstavlja pomemben korak k razvoju nevirusnih strategij za genetsko inženirstvo celic T v okviru celične imunoterapije raka. 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. Imunoterapija raka celična terapija CRISPR/Cas celična linija Jurkat TCR Cancer immunotherapy cell therapy CRISPR/Cas Jurkat cell line TCR true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-25 08:25:00 2025-09-25 08:25:04 2025-11-19 14:05:17 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-09-23 23081 257832451 Kunej_Klemen_-_Nevirusno_urejanje_genoma_T-celicne_linije_Jurkat_za_izbitje_tarcnega_gena_TRAC_i.pdf Kunej_Klemen_-_Nevirusno_urejanje_genoma_T-celicne_linije_Jurkat_za_izbitje_tarcnega_gena_TRAC_i.pdf 1 EE95D24681FDA7FA7EE914A2DA031465 a868a4a419ca2944a54463884ecedf9bb28f1c65f9fa000ae9d48f4e73930c9b 30046dac-99d8-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=218636 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0