20.500.12556/RUL-186240 Uporaba tehnologije CRISPR-dCas9 za spreminjanje izražanja genov preko acetilacije histonov pri kvasovki Saccharomyces cerevisiae Utilization of CRISPR-dCas technology for modulating gene expression through histone acetylation in the yeast Saccharomyces cerevisiae V magistrskem delu smo z uporabo tehnologije CRISPR-dCas9, združene s histonskimi modifikatorji Gcn5, Sir2 in Rpd3, preučevali vpliv izražanja peroksisomskih genov FOX2, CAT2 in POX1 pri kvasovki Saccharomyces cerevisiae. Namen raziskave je bil ugotoviti, ali lahko lokalna acetilacija oziroma deacetilacija histonov v promotorskih regijah spremeni stopnjo transkripcije izbranih genov ter ali se takšna sprememba odrazi v fenotipu na ravni hitrosti rasti. Po konstrukciji plazmidov in transformaciji sevov kvasovke smo delovanje posameznih fuzijskih kompleksov ovrednotili fenotipsko s kapljičnimi testi rasti na različnih virih ogljika ter z izolacijo celokupne RNA iz celic kvasovke, gojenih v bogatem gojišču z glukozo, z uporabo metode RT-qPCR. Rezultati so pokazali, da kompleks dCas9-Gcn5 poveča izražanje FOX2, vendar se ta aktivacija ne odrazi v izboljšani rasti, kar kaže na presnovno neravnovesje v odsotnosti indukcijskih signalov za β-oksidacijo. Konstrukta dCas9-Sir2 in dCas9-Rpd3 sta pričakovano znižala izražanje CAT2, medtem ko je presenetljivo tudi kompleks dCas9-Gcn5 znižal izražanje CAT2, kar nakazuje močan vpliv glukozne represije, ki lahko nadvlada lokalne epigenetske signale. Izražanje POX1 je ostalo nizko ne glede na epigenetsko modulacijo, kar nakazuje močno intrinzično uravnavanje izražanje tega gena. Skupno ugotovitve kažejo, da epigenetska modulacija učinkuje le, kadar je skladna s presnovnim stanjem celice, in poudarjajo potrebo po nadaljnjem preučevanju teh pristopov v pogojih, kjer so dolgoverižne maščobne kisline edini ali primarni vir ogljika. In this master’s thesis, we investigated the effects of CRISPR-dCas9 technology fused with the histone modifiers Gcn5, Sir2, and Rpd3 on the expression of the peroxisomal genes FOX2, CAT2, and POX1 in the yeast Saccharomyces cerevisiae. The aim of the study was to determine whether local histone acetylation or deacetylation in promoter regions can alter the transcription rate of selected genes and whether such changes are reflected at the phenotypic level as a change in growth rate. Following plasmid construction and yeast transformation, the activity of individual fusion complexes was evaluated phenotypically using spot growth assays on different carbon sources and by isolating total yeast RNA from yeast cells grown in a rich medium with glucose, followed by gene expression measurements using the RT-qPCR method. The results showed that the dCas9-Gcn5 complex increased FOX2 expression; however, this activation was not reflected in improved growth, indicating metabolic imbalance in the absence of induction signals for β-oxidation. The dCas9-Sir2 and dCas9-Rpd3 constructs, as expected, reduced CAT2 expression, while, surprisingly, the dCas9- Gcn5 complex also reduced CAT2 expression, suggesting a strong influence of glucose repression that can override local epigenetic signals. POX1 expression remained low regardless of epigenetic modulation, indicating its strict metabolic regulation. Overall, the findings demonstrate that epigenetic modulation is effective only when it is aligned with the metabolic state of the cell and highlight the need for further investigation of these approaches under conditions where long-chain fatty acids are the sole or primary carbon source. CRISPR-dCas9 epigenetika acetilacija histonov deacetilacija histonov izražanje genov Saccharomyces cerevisiae peroksisomski geni B-oksidacija CRISPR-dCas9 epigenetika acetilacija histonov deacetilacija histonov izražanje genov Saccharomyces cerevisiae peroksisomski geni β-oksidacija true false false [N. Golja] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-08-29 07:17:02 2026-08-29 07:17:37 2026-08-31 10:01:45 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 602.3:582.282.23(043.2) 286178 289411843 59871.pdf 59871.pdf 1 F7491A0ABF06D31A826AD5159328E38E 2721dc304900a1dd7a30cefcfc8d3938eaf97a275da6a17fff4101c380b20fc3 a7d1d3a8-a368-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=245378 Biotehniška fakulteta 0 0 0