Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus

master's thesis

• Title (english): Effect of sgRNA length on efficiency and specificity of Staphylococcus aureus Cas9 protein
• Author: Stjepan Rakitić
• Mentor: Aleksandar Vojta (mentor)
• Committee member: Aleksandar Vojta (predsjednik povjerenstva)
• Committee member: Damjan Franjević (član povjerenstva)
• Committee member: Toni Nikolić (član povjerenstva)
• Committee member: Vlatka Zoldoš (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-07-10, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Sustav CRISPR/Cas9, adaptivni imunosni sustav prokariota, iskorišten je kao metoda za ciljano uređivanje genoma. Cas9 inaktiviran mutacijama (dCas9) može se koristiti za navođenje efektorskih domena. Ciljani gen moguće je metilirati korištenjem fuzije dCas9-DNMT3A, ili demetilirati sa dCas9-TET1. Problem ovakvih sustava je nespecifična aktivnost izvan ciljnih lokusa, što se može smanjiti korištenjem skraćenih sgRNA. Cilj istraživanja bio je utvrditi utjecaj različitih duljina sgRNA na specifičnost sustava za ciljanu promjenu statusa metilacije DNA. Ekspresijske kazete za sgRNA klonirane su u vektore za koekspresiju dCas9-DNMT3A, odnosno dCas9-TET1, kojima je transfecirana stanična linija HEK293. Nakon transfekcije stanična DNA je izolirana te pirosekvencirana nakon bisulfitne konverzije radi određivanja metilacijskog statusa. Skraćivanje molekula sgRNA nije dovelo do statistički značajnog smanjenja nespecifične aktivnosti. Nije utvrđen veći utjecaj skraćivanja molekula sgRNA na nespecifičnu aktivnost u odnosu na specifičnu aktivnost. Optimalna duljina molekule sgRNA za ciljanje gena LAMB1 i fragment F3 gena BACH2 je 21 nukleotid te 20 nukleotida za fragment F4 gena BACH2 i fragment A1 promotora gena MGAT3.

Abstract (english)

The CRISPR/Cas9 system, originally discovered as adaptive bacterial immunity, had been repurposed as a tool for genome editing. Cas9 rendered inactive by mutations (dCas9) can be used for guiding effector domains. Targeted gene can be methylated using dCas9-DNMT3A fusion, or demethylated using dCas9-TET1. Problem such systems have is non-specific activity beyond targeted genes, which can be reduced using truncated sgRNA. Aim of the study was to determine the effect of different sgRNA lengths on specificity of systems for targeted changing of DNA methylation status. sgRNA expression cassettes were cloned into vectors coexpressing dCas9-DNMT3A and dCas9-TET1 respectively, which were used for transfecting HEK293 cell line. After the transfection DNA was isolated and pyrosequenced after bisulphite treatment to determine methylation status. Truncating sgRNAs didn't result in statistically significant reduction of non-specific activity. Increased influence of truncating sgRNAs on non-specific activity compared to specific activity was not determined. Optimal sgRNA length for targeting F4 fragment of LAMB1 gene and F3 fragment of BACH2 gene is 21 nucleotides, and 20 nucleotides for fragment F4 of BACH2 gene and for A1 fragment of the MGAT3 gene promotor.

• Keywords: dCas9; DNMT3A; TET1; modificiranje epigenoma; HEK293; metilacija
• Keywords (english): dCas9; DNMT3A; TET1; epigenome editing; HEK293; methylation
• Language: croatian
• URN:NBN: urn:nbn:hr:217:758236
• 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
• Created on: 2018-08-29 09:03:25