Korištenje CRISPR/Cas9 metode za inaktivaciju puta DNA popravka izrezivanjem nukleotida (engl. Nucleotide Excision Repair) u zebrici (Danio Rerio)

undergraduate thesis

• Title (english): Use of CRISPR/Cas9 method for inactivation of DNA repair with Nucleotide Excision Repair in zebrafish (Danio rerio)
• Author: Lucija Mance
• Mentor: Igor Stuparević (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2020-07-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Cilj ovog rada bio je konstruirati RNA vodilju (engl. single guide RNA, sgRNA) za CRISPR/Cas9 inaktivaciju xpa gena u ribi zebrici (lat. Danio rerio), sa svrhom inaktivacije puta DNA popravka izrezivanjem nukleotida (engl. Nucleotide Excision Repair, NER), kako bi se utvrdila uloga NER puta popravka u DPC popravku (engl. DNA-protein crosslink repair, DPC repair) in vivo u modelnom organizmu. Dizajniran je DNA kalup za sintezu sgRNA za xpa in silico te in vitro sljepljivanjem DNA proba. Kalup je umnožen lančanom reakcijom polimeraze (engl. Polymerase Chain Reaction, PCR) te vizualiziran DNA elektroforezom u agaroznom gelu. PCR amplikon služio je kao kalup za sintezu sgRNA in vitro transkripcijom. Kao provjera in vitro transkripcije provedena je RNA elektroforeza u agaroznom gelu. Sintetizirana sgRNA visoke je kvalitete i koncentracije. Uspješnost djelovanja sgRNA dalje se provjerava konstrukcijom Cas9/sgRNA kompleksa, mikroinjektiranjem embrija zebrice i praćenjem uvedenih promjena genotipizacijom pomoću HRMA metode (engl. High Resolution Melting Analysis).

Abstract (english)

The aim of this paper was to create single guide RNA (sgRNA) for CRISR/Cas9 xpa gene inactivation in zebrafish (lat. Danio rerio) and consequently Nucleotide Excision Repair (NER) inactivation. The purpose of inactivation is to investigate the role of NER in DNA-protein crosslink repair (DPC repair). Firstly, DNA template for xpa sgRNA was designed in silico. DNA template synthesis in vitro included linking of DNA probes and amplification of the template by polymerase chain reaction (PCR) and its visualisation using DNA electrophoresis in agarose gel. PCR amplicon was used as a template for sgRNA synthesis using in vitro transcription. Presence of RNA was confirmed by RNA electrophoresis in agarose gel. Synthesized sgRNA was of high quality and concentration. Subsequently, efficiency of sgRNA synthesis will be checked after Cas9/sgRNA complex construction and microinjection in zebrafish embryos followed by genotyping using HRMA method.

• Keywords: CRISPR/Cas9; DPC; NER; xpa; zebrica
• Keywords (english): CRISPR/Cas9; DPC; NER; xpa; zebrafish
• Language: croatian
• URN:NBN: urn:nbn:hr:159:809703
• Study programme: Title: Biotechnology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-11-10 14:40:38