diplomsko delo
Ana Potočnik (Author), Jernej Ogorevc (Mentor)

Abstract

Sistem CRISPR/Cas9 je uporabno orodje za urejanje genoma in vključuje enoverižno vodilno RNA (sgRNA), ki je komplementarna tarčnemu zaporedju, ter endonukleazo Cas9. Z vnosom mutacij v katalitična mesta Cas9 so raziskovalci ustvarili »mrtvi« Cas9 (dCas9), ki nima endonukleazne aktivnosti. Na dCas9 lahko pripojimo različne transkripcijske dejavnike, s katerimi lahko uravnavamo prepisovanje in posledično tudi izražanje genov. Glede na uporabo različnih transkripcijskih dejavnikov (npr. KRAB, VP160, VPR) in zaporedij sgRNA ločimo različne sisteme, ki so namenjeni aktivaciji (CRISPRa) ali interferenci (CRISPRi) izražanja genov in se med seboj razlikujejo po učinkovitosti. V diplomskem delu smo opisali značilnosti različnih sistemov za regulacijo prepisovanja, fuziji dCas9-VP160 in dCas9-VPR ter štiri različne sgRNA pa smo tudi praktično preizkusili za povečanje izražanja gena TLR10 v celični liniji A549. Po transfekciji celične linije s plazmidi, ki so vsebovali različne sgRNA in aktivatorske fuzije z dCas9, smo iz celic izolirali celokupno RNA. Relativno kvantifikacijo povečanja izražanja tarčnega gena smo določili z metodo RT-qPCR. Rezultati kažejo, da sta oba sistema učinkovita pri aktivaciji izražanja gena TLR10. Potrdili smo tudi, da je sistem CRISPR/dCas9-VPR učinkovitejši od sistema CRISPR/dCas9-VP160, saj prepisovanje gena poveča za približno 15-krat, medtem ko drugi le za največ 1,5-krat. Na raven povečanja izražanja je pomembno vplivalo zaporedje sgRNA. Sistemi CRISPR/dCas9 predstavljajo perspektivno tehnologijo za uravnavanje izražanja genov z najrazličnejšimi načini uporabe na področju funkcijske genomike.

Keywords

CRISPR;dCas9;sistem CRISPR/Cas;sgRNA;izražanje genov;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [A. Potočnik]
UDC: 577.21(043.2)
COBISS: 82928131 Link will open in a new window
Views: 216
Downloads: 42
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Other data

Secondary language: English
Secondary title: Use of CRISPR/dCas9 systems for regulation of TLR10 gene expression in lung epithelial cell line A549
Secondary abstract: 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.
Secondary keywords: CRISPR;dCas9;sgRNA;gene expression;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 35 str.
ID: 13377258