Izrada modularnog sustava CRISPR/dCas9 upotrebom ortolognih proteina Cas9 i njegova primjena u epigenetičkim manipulacijama genskih lokusa

doctoral thesis

• Title (english): design of CRISPR/dCas9 modular system using orthogonal Cas9 proteins and its application in epigenetic manipulation of gene loci
• Author: Goran Josipović
• Mentor: Vlatka Zoldoš (mentor)
• Committee member: Aleksandar Vojta (predsjednik povjerenstva)
• Committee member: Mirjana Pavlica (član povjerenstva)
• Committee member: Srećko Gajović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-05-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

Otkrivanje uzročno posljedičnih veza između epigenetičkih oznaka u kromatinu i regulacije
ekspresije gena omogućeno je primjenom molekularnih alata koji mogu ciljano mijenjati
epigenom. U ovom istraživanju izradio sam modularni sustav temeljen na CRISPR/dCas9
metodologiji za ciljanu epigenetičku manipulaciju i direktnu regulaciju ekspresije gena,
sastavljen od glavnih modula: promotora RNA polimeraze II, ortologa dCas9, efektorske
domene, selekcijskog biljega, te modula za primanje više molekula sgRNA potrebnih za
navođenje konstrukta na ciljno mjesto u genomu. Ciljanjem parova genskih lokusa BACH2-
HNF1A i IL6ST-MGAT3 s fuzijama antagonističkog djelovanja (metilacija vs. demetilacija
DNA), uz korištenje dva ortologa dSpCas9 i dSaCas9 u stanicama HEK293, pokazao sam da
je moguće promijeniti stupanj metilacije DNA kojeg prati promjena u ekspresiji tih gena.
Također sam pokazao da epigenetičkom manipulacijom para gena MGAT3-HNF1A, u
stanicama BG1, mogu promijeniti glikanski fenotip. Istovremenim korištenjem fuzijskih
konstrukata TET1-dSaCas9 i VPR-dSpCas9 postigao sam sinergistički učinak na ekspresiju
gena HNF1A koji je postojan kroz duži vremenski period. Dodatno sam unaprijedio modularni
sustav u svrhu smanjenja nespecifičnog učinka.

Abstract (english)

Establishing a causal relationship between epigenetic marks in chromatin and the regulation of
gene expression is made possible by the use of molecular tools that can specifically alter the
epigenome. In this study, I developed a modular system based on the CRISPR/dCas9
methodology for targeted epigenetic manipulation and direct regulation of gene expression,
consisting of major modules: RNA polymerase II promoter, dCas9 ortholog, effector domain,
selection marker and module that can receive multiple sgRNA molecules to guide the construct
in the genome. By targeting BACH2-HNF1A and IL6ST-MGAT3 gene pairs with fusions
showing antagonistic functionalities (DNA methylation vs. demethylation), using two orthologs
dSpCas9 and dSaCas9 in HEK293 cells, I showed that it is possible to change the DNA
methylation accompanied by a change in gene expression. I also showed that by epigenetic
manipulation of MGAT3-HNF1A gene pair, in BG1 cells, the glycan phenotype can be altered.
By simultaneously using the fusion constructs TET1-dSaCas9 and VPR-dSpCas9, I achieved a
synergistic effect on HNF1A gene expression that is stable over a longer period of time. I further
improved the modular system in order to reduce the off-target effect.

• Keywords: sustav CRISPR/dCas9; ortologni proteini Cas9; ciljana manipulacija epigenoma; regulacija ekspresije gena; nespecifični učinak sustava CRISPR/dCas9
• Keywords (english): CRISPR/dCas9 system; Cas9 orthologous proteins; targeted epigenome editing; direct regulation of gene expression; CRISPR/dCas9 off-target effect
• Language: croatian
• URN:NBN: urn:nbn:hr:217:756265
• Promotion: 2021
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-07-14 12:23:50