Ciljana mutageneza gena SPRTN i Tdp2a u zebrici (Danio rerio) pomoću sustava CRISPR

master's thesis

• Title (english): Targeted mutagenesis of SPRTN and Tdp2a genes in zebrafish (Danio rerio) using the CRISPR system
• Author: Matea Eršetić
• Mentor: Marta Popović (mentor)
• Mentor: Ivana Ivančić Baće (sumentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Ivana Šola (član povjerenstva)
• Committee member: Dubravka Hranilović (član povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-09-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Unakrsna vezanja DNA-protein nastaju uspostavom ireverzibilnih kovalentnih veza između
DNA i proteina, a njihovo je uklanjanje neophodno za očuvanje integriteta staničnog genoma.
Metaloproteaza Sprtn sudjeluje u proteolitičkoj razgradnji adukata histona i topoizomeraza, a
nastali peptidni ostatci topoizomeraze II supstrat su za tirozil-DNA fosfodiesterazu 2 (Tdp2).
Mutacije navedenih proteina smanjuju sposobnost popravka oštećenja DNA, a poznata je
mutacija Y117C koja uzrokuje sindrom Rujis-Aalfs u ljudi, kojeg karakteriziraju preuranjeno
starenje i razvoj hepatocelularnog karcinoma u mladoj dobi. Uvođenjem mutacije L147A u
katalitičku regiju gena sprtn ribe zebrice (Danio rerio) pomoću sustava CRISPR/Cas12a,
izradila sam animalni model sa smanjenom efikasnošću popravka unakrsnih vezanja DNAprotein za proučavanje funkcije proteina Sprtn. Također, selektirala sam dva utemeljitelja s
pomakom okvira čitanja u genu tdp2a zebrice nakon primjene sustava CRISPR/Cas9 i uzgojila
njihovo potomstvo. Uzgoj homozigotnih linija za navedene mutacije i mogućnost stvaranja
dvostrukih mutanata poboljšat će razumijevanje uloga proteina Tdp2a i Sprtn u popravku
oštećenja DNA te imati potencijalne primjene u liječenju bolesti čovjeka uzrokovanih
mutacijama u navedenim genima.

Abstract (english)

DNA-protein crosslinks occur by establishing irreversible covalent bonds between DNA and
proteins, and their removal is necessary to preserve the integrity of the cellular genome. The
metalloprotease Sprtn participates in the proteolytic degradation of histone and topoisomerase
adducts, and the resulting peptide residues of topoisomerase II are substrates for tyrosyl-DNA
phosphodiesterase 2 (Tdp2). Mutations in these proteins reduce the ability to repair DNA
damage, and the Y117C mutation is known to cause Rujis-Aalfs syndrome in humans, which
is characterized by premature aging and the development of hepatocellular carcinoma at a
young age. By introducing the L147A mutation into the catalytic region of the zebrafish (Danio
rerio) sprtn gene using the CRISPR/Cas12a system, I developed an animal model with reduced
DNA-protein crosslink repair efficiency to study Sprtn protein function. Also, I selected two
founders with a frameshift in the zebrafish tdp2a gene after applying the CRISPR/Cas9 system
and bred their offspring. The cultivation of homozygous lines for these mutations and the
ability to form double mutants will improve understanding of the roles of Tdp2a and Sprtn
proteins in DNA damage repair and have potential applications in the treatment of human
diseases caused by mutations in these genes.

• Keywords: unakrsna vezanja DNA-protein; popravak oštećenja DNA; animalni model
• Keywords (english): DNA-protein crosslinks; DNA damage repair; animal model
• Language: croatian
• URN:NBN: urn:nbn:hr:217:045651
• 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; Embargo expiration date: 2023-09-27
• Created on: 2022-01-25 12:33:35