The CRISPR/Cas9 system is a useful tool for genome editing, consisting of single-stranded RNA (sgRNA) complementary to target sequence, and Cas9 endonuclease. Dead Cas9 (dCas9) which lacks endonuclease activity was created by mutating catalytic sites of Cas9 protein. Different transcription factors may be fused to dCas9 to regulate gene transcription. Based on fused transcription regulators (e.g. KRAB, VP160, VPR) and sgRNA, scaffold sequences are available for CRISPR activation (CRIPSRa) and interference (CRISPRi), which vary in gene regulation efficiency. In this thesis, we provide description of different systems. Additionally, we used dCas9-fused VP160 and VPR activators to induce transcription of the TLR10 in lung epithelial cell line A549. After transfection with plasmids, containing different sgRNAs and dCas9 activation fusions, total RNA was isolated from the cells. Relative transcription of the target gene was determined by RT-qPCR. Our results show that both fusions were able to induce TLR10 transcription, however VPR turned out to be a much more potent activator of transcription than VP160. Activation was highly dependent on the sgRNA sequence used. CRISPR/dCas9 systems are a promising technology for gene regulation with versatile applications in the field of functional genomics.
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