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Genetic code expansion and dCas9-DAXXHBD as synthetic routes for epigenome editing
University of Skövde, School of Health Sciences.
2020 (English)Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Epigenome editing has become a promising new tool to study the effects of singular histone modifications on the transcriptional activity of mammalian cells. Although many different approaches have been developed during the recent years many approaches used dCas9 fused epigenetic effector proteins to deposit the epigenetic mark at target sites. One major concern of these methods are the extensive and uncontrolled off target effects on chromatin associated proteins which can bias the interpretation of such experiments. This project validates a recently developed dCas9- DAXXHBD fusion protein which would allow the site-specific incorporation of the replication independent H3.3 variant and the use of genetic code expansion to generate a pool of site specifically acylated histones. Both approaches in combinations can provide a novel technique to deposit pre modified H3.3 at specific genomic loci. By using an evolved PylRS pair, it is shown that one stable cell line can give raise to a set of site- specifically acylated H3.3 molecules. However, it is unclear if these histones can be successfully incorporated into chromatin. Immunoblotting and microscopy studies showed that the dCas9-DAXXHBD construct can be successfully expressed upon amber suppression in stable cell lines. However, the obtained data shows that only a small fraction of the construct translocate into the nucleus. The preformed ChIP- qPCR analysis aimed to show the deposition activity of the construct at targeted genomic loci but failed to proof the concept due to technical reasons and the probable low nuclear concentration of the construct. Therefore, both techniques must be further verified in order to determine, if they could provide a reliable fundament of the next generation of epigenetic editors.

Place, publisher, year, edition, pages
2020. , p. 41
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:his:diva-19051OAI: oai:DiVA.org:his-19051DiVA, id: diva2:1466504
External cooperation
Karolinska Institutet; Science for Life Laboratory: ElsässerLab
Subject / course
Biomedicine/Medical Science
Educational program
Biomedicine - Study Programme
Supervisors
Examiners
Available from: 2020-09-11 Created: 2020-09-11 Last updated: 2020-09-11Bibliographically approved

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