CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION

doctoral thesis

• Title (croatian): STANIČNE LINIJE S INTEGRIRANIM CRISPRa I CRISPRi SUSTAVOM I NJIHOVA PRIMJENA U ANALIZI GLIKOZILACIJE IMUNOGLOBULINA G
• Author: Anika Mijakovac
• Mentor: Aleksandar Vojta (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Ivana Ivančić Baće (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-03-20, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

Mechanisms regulating immunoglobulin G (IgG) glycosylation are largely unknown. To
perform functional validation of genes implicated in IgG glycosylation we developed the
HEK293-F system. Utilizing transposons, we integrated dCas9-VPR/dCas9-KRAB expression
cassettes in the genome of FreeStyle™ 293-F cells for targeted manipulation
(CRISPRa/CRISPRi) of selected genes. Transfection of HEK293-F cells with IgG-gRNAbearing plasmid enabled simultaneous analysis of gene expression and IgG glycome. Utilizing
this system, we validated the role of the SPPL3 locus in IgG sialylation. Activation of estrogenregulated genes, RUNX3 and SPINK4, decreased the galactosylated IgG glycoforms, a
phenotype characteristic of menopause and aging. To further examine the magnitude of genetic
influences on IgG glycosylation in aging we performed a heritability analysis on the glycan
clock marker of biological age. Heritability of the glycan clock, when corrected for the age of
the individuals, proved to be high (~71%). This served to demonstrate the necessity to develop
new technologies able to dissect the observed genetic factors regulating IgG glycosylation
associated not only with disease but also with aging.

Abstract (croatian)

Regulatorni mehanizmi glikozilacije imunoglobulina G (IgG) većinom su nepoznati. Kako
bismo proveli funkcionalnu potvrdu uloge gena asociranih s IgG glikozilacijom razvili smo
sustav HEK293-F. Korištenjem transpozona, ugradili smo kazete dCas9-VPR/dCas9-KRAB u
genom stanica FreeStyle™ 293-F za ciljanu manipulaciju (CRISPRa/CRISPRi) odabranih
gena. Transfekcija stanica HEK293-F s plazmidom IgG-gRNA omogućila nam je istovremenu
analizu ekspresije gena i IgG glikoma. Korištenjem ovog sustava potvrdili smo ulogu lokusa
SPPL3 u IgG sijalinizaciji. Aktiviranjem gena reguliranih estrogenom, RUNX3 i SPINK4, došlo
je do smanjenja galaktoziliranih IgG glikoformi odnosno pojave fenotipa karakterističnog za
menopauzu i starenje. Kako bismo dodatno istražili opseg genetičkih utjecaja na IgG
glikozilaciju tijekom starenja, proveli smo analizu nasljednosti markera biološke dobi –
glikanskog sata. Nasljednost glikanskog sata se pokazala visokom nakon korekcije za dob
sudionika (~71%) čime smo pokazali kako je neophodno razvijati nove tehnologije koje će
razjasniti genetičke faktore uključene u regulaciju glikozilacije IgG-a, povezane ne samo s
bolestima već i sa starenjem.

• Keywords: glycosylation; immunoglobulin G; CRISPR/dCas9; gene regulation; heritability
• Keywords (croatian): glikozilacija; imunoglobulin G; CRISPR/dCas9; regulacija gena; nasljednost
• Language: english
• URN:NBN: urn:nbn:hr:217:090828
• Promotion: 2023
• 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: 2023-04-03 09:33:13