Konstrukcija modularnih alata KDM5A-dCas9 i PAD4-dCas9 u svrhu ciljane manipulacije ekspresije gena

master's thesis

• Title (english): Construction of KDM5A-dCas9 and PAD4-dCas9 modular tools for targeted manipulation of gene expression
• Author: Ivana Keršić
• Mentor: Aleksandar Vojta (mentor)
• Committee member: Aleksandar Vojta (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Committee member: Anamaria Štambuk (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-07-14, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sustav CRISPR/Cas modificiran je uvođenjem deaktivirane endonukleaze Cas9 (dCas9)
na koju se mogu vezati katalitičke domene efektorskog proteina, omogućujući ciljanu
manipulaciju genoma i epigenoma. Daljnjom modifikacijom, nespecifična nukleaza dCas9 navodi
se na više ciljanih područja u genomu pomoću više različitih molekula sgRNA. Fuzija KDM5A s
dCas9 omogućuje ciljanu demetilaciju histonske pozicije H3K4 čime dovodi do smanjenja genske
ekspresije, a fuzija dCas9 s PAD4 omogućuje ciljanu citrulinaciju histona čime dovodi do
povećanja genske ekspresije. Cilj ovog istraživanja bio je konstruirati fuzijske konstrukte
KDM5A-dCas9 i PAD4-dCas9 u sklopu modularnog CRISPR/dCas9 sustava s ciljem proučavanja
utjecaja na ekspresiju ciljanih gena TWIST1, KLF8, SNAI1 i ZEB1. Uspješno su dobiveni
katalitički aktivni konstrukti za ciljanu manipulaciju ekspresije gena kojima su transfecirane
stanice HepG2, kao i kontrolni katalitički inaktivni i non-target konstrukti. Izolirana RNA
podvrgnuta je qRT-PCR-u, a rezultati pokazuju da nema statistički značajnog smanjenja ekspresije
gena SNAI1 i ZEB1 u stanicama transfeciranim plazmidnim konstruktima s aktivnim KDM5A,
kao niti u kontrolnim uzorcima. Statistički značajno povećanje ekspresije gena KLF8 u
stanicama transfeciranim plazmidnim konstruktima s aktivnim i inaktivnim PAD4 vidljivo je osam
dana nakon transfekcije, no ne i 12 dana nakon. Vrijednosti Ct nisu dobivene za gen TWIST1 stoga
njegova ekspresija nije mogla biti analizirana.

Abstract (english)

The CRISPR/Cas system is modified by introducing a deactivated Cas9 endonuclease to
which effector protein catalytic domains can be bound, allowing for targeted genome and
epigenome manipulation. With further modification, the non-specific nuclease dCas9 can be
guided to multiple genome sites using mutliple different sgRNA molecules. Fusion of KDM5A
with dCas9 allows for targeted demethylation of histone position H3K4 leading to decreased gene
expression, while fusion of PAD4 with dCas9 allows for targeted histone citrullination leading to
increased gene expression. The aim of this research was to construct fusion constructs KDM5AdCas9 and PAD4-dCas9 in a modular CRISPR/dCas9 system with the goal of observing the effect
on expression of targeted genes TWIST1, KLF8, SNAI1 i ZEB1. Catalytically active constructs for
targeted manipulation of gene expression were successfully constructed as were inactive and nontarget constructs for control purposes, and used to transfect HepG2 cells. Isolated RNA was
subjected to qRT-PCR. Results show no significantly lowered expression of SNAI1 and ZEB1
genes in cells transfected with active KDM5A construct nor in control samples. Significantly
increased expression of KLF8 gene is present in cells transfected with active and inactive PAD4
contruct 8 days after transfection, but not 12 day after. Ct values for TWIST1 genes were not
obtained therefore its expression could not be analyzed.

• Keywords: epigenetika; ciljano modificiranje epigenoma; dCas9; KDM5A; PAD4; ekspresija
• Keywords (english): epigenetics; targeted epigenome editing; dCas9; KDM5A; PAD4; expression
• Language: croatian
• URN:NBN: urn:nbn:hr:217:006180
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-07-22 10:39:58