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.
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