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doctoral thesis
Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression
Zagreb: University of Zagreb, Faculty of Science, 2019. urn:nbn:hr:217:976868
Tadić, Vanja
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Tadić, V. (2019). Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression (Doctoral thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:976868

Tadić, Vanja. "Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression." Doctoral thesis, University of Zagreb, Faculty of Science, 2019. https://urn.nsk.hr/urn:nbn:hr:217:976868

Tadić, Vanja. "Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression." Doctoral thesis, University of Zagreb, Faculty of Science, 2019. https://urn.nsk.hr/urn:nbn:hr:217:976868

Tadić, V. (2019). 'Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression', Doctoral thesis, University of Zagreb, Faculty of Science, accessed 22 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:976868

Tadić V. Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression [Doctoral thesis]. Zagreb: University of Zagreb, Faculty of Science; 2019 [cited 2024 November 22] Available at: https://urn.nsk.hr/urn:nbn:hr:217:976868

V. Tadić, "Construction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression", Doctoral thesis, University of Zagreb, Faculty of Science, Zagreb, 2019. Available at: https://urn.nsk.hr/urn:nbn:hr:217:976868

Metadata
TitleConstruction of molecular tools based on CRISPR/Cas9 system for epigenetic modulation of gene expression
Title (croatian)Konstrukcija molekularnih alata temeljenih na sustavu CRISPR/Cas9 za epigenetičku modulaciju ekspresije gena
AuthorVanja Tadić
MentorVlatka Zoldoš (mentor)
Committee memberAleksandar Vojta (predsjednik povjerenstva)
Committee memberSrećko Gajović (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2019-01-28, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Universal decimal classification
(UDC)		577 - Biochemistry. Molecular biology. Biophysics
Abstract
Repurposing of platforms for targeted genome engineering, zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs) and especially CRISPR/Cas9 system, has enabled targeted manipulation of epigenetic marks and new insights into epigenetic regulatory mechanisms. In this work, I have fused the catalytic domain of human TET1 (Ten-Eleven Translocation 1) protein Cterminally to the catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes to create the dCas9- TET1 molecular tool. I have also tested the activity of N-terminal fusion of TET1-dCas9. The TET1 protein is a methylcytosine dioxygenase involved in active demethylation of 5-methylctosines to 5- hydroxymethylcytosines. In context of gene expression, methylated CpG dinucleotides within gene promoters are usually associated with gene silencing, and unmethylated CpGs with active gene transcription. Constructed molecular tools were successfully used for targeted CpG demethylation in promoter regions of two candidate genes, MGAT3 and LAMB1. I also showed that following demethylation the MGAT3 gene changed expression level. I have compared MGAT3 gene activation rates achieved following demetlylation using dCas9-TET1 with a more conservative approach of VPR-dCas9 mediated direct activation. For this approach, I used an N-terminal fusion of VPR activation domain and dCas9 from Staphylococcus aureus (dSaCas9).
Abstract (croatian)
Prenamjene alata za genetičko inženjerstvo, baziranih na zinc finger nukleazama (ZFN), transcription activator-like effector nukleazama (TALEN) te posebno sustavu CRISPR/Cas9, pružile su mogućnost ciljanog modificiranja epigenoma i time razjašnjavanje uloge epigenetičkih modifikacija u regulaciji ekspresije gena. U ovom radu napravila sam C-terminalnu fuziju katalitičke domene proteina TET1 (Ten-Eleven Translocation 1) i katalitički inaktivne nukleaze Cas9 (dCas9) izolirane iz vrste Streptococcus pyogenes i konstruirala dCas9-TET1 molekularni alat za ciljanu demetilaciju DNA. Testirala sam i aktivnost N-terminalne fuzije TET1-dCas9. Protein TET1 katalizira pretvorbu 5- metilcitozina u 5-hidroksimetilcitozin, što predstavlja prvi korak u aktivnoj demetilaciji DNA. Korelacijske analize povezuju hipermetilirane CpG dinukleotide u promotorima gena s represijom transkripcije, a hipometilirane CpG dinukleotide s aktivnom transkripcijom. Konstruirani alati uspješno su upotrijebljeni za ciljanu demetilaciju CpG dinukleotida unutar promotora kandidat gena MGAT3 i LAMB1. Pokazala sam da je nakon demetilacije ciljanih CpG mjesta u promotoru gena MGAT3 porasla razina transkripata ovoga gena. Usporedila sam efekt aktivacije gena MGAT3 postignut ciljanom demetilacijom dCas9-TET1 alatom s konzervativnijim pristupom direktne aktivacije pomoću VPR-dCas9 alata. U ovu svrhu upotrijebila sam N-terminalnu fuziju aktivacijske domene VPR i dCas9 iz vrste Staphylococcus aureus (dSaCas9).
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:217:976868
Study programmeTitle: 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 resourceText
File originBorn digital
Access conditionsOpen access
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Created on2019-02-05 09:56:18