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master's thesis
Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus
Zagreb: University of Zagreb, Faculty of Science, 2018. urn:nbn:hr:217:758236
Rakitić, Stjepan
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

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Rakitić, S. (2018). Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:758236

Rakitić, Stjepan. "Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:758236

Rakitić, Stjepan. "Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:758236

Rakitić, S. (2018). 'Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus', Master's thesis, University of Zagreb, Faculty of Science, accessed 04 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:758236

Rakitić S. Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2018 [cited 2024 November 04] Available at: https://urn.nsk.hr/urn:nbn:hr:217:758236

S. Rakitić, "Utjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2018. Available at: https://urn.nsk.hr/urn:nbn:hr:217:758236

Metadata
TitleUtjecaj duljine molekule sgRNA na učinkovitost i specifičnost proteina Cas9 iz vrste Staphylococcus aureus
Title (english)Effect of sgRNA length on efficiency and specificity of Staphylococcus aureus Cas9 protein
AuthorStjepan Rakitić
MentorAleksandar Vojta (mentor)
Committee memberAleksandar Vojta (predsjednik povjerenstva)
Committee memberDamjan Franjević (član povjerenstva)
Committee memberToni Nikolić (član povjerenstva)
Committee memberVlatka Zoldoš (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2018-07-10, 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
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:758236
Study programmeTitle: Molecular Biology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2018-08-29 09:03:25