Construction of KDM5A-dCas9 and PAD4-dCas9 modular tools for targeted manipulation of gene expression

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.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