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Zitiere als: Stachler, Aris-Edda (2017): Das CRISPR-Cas-System von Haloferax volcanii: CRISPRi und Autoimmunität. Open Access Repositorium der Universität Ulm. Dissertation. http://dx.doi.org/10.18725/OPARU-4511

AutorStachler, Aris-Eddadc.contributor.author
In OPARU verfügbar seit2017-09-13T08:01:52Zdc.date.available
Jahr der Erstellung2017dc.date.created
Datum der Erstveröffentlichung2017-09-13dc.date.issued
ZusammenfassungThe CRISPR-Cas system is a prokaryotic adaptive, heritable immune system. The crRNA, which orchestrates the sequence-specific binding of a target sequence, constitutes a key component of the defence response. Employing mutational analyses and a Cas6b-independent system for crRNA biogenesis, important parts of the 5‘ and 3‘ handles could be identified. Results points towards an essential role of the 5‘ handle for interference, whereas the 3‘ handle is completely dispensable. Within the frame of another project the effects of CRISPR-Cas-mediated autoimmunity were analyzed in H. volcanii. Even when the expression of endogenous crRNAs was inhibited and only a crRNA against genomic DNA was present, the observed toxicity was moderate. While H. volcanii cells were being subjected to an autoimmune reaction, they showed impaired fitness, but by introducing deletions in the region of the target sequence, self-targeting was quickly abolished. In a wildtype strain synthetising all endogenous crRNAs, the expression of a crRNA against genomic DNA showed no detrimental effects. The polyploidy of H. volcanii can presumably act as a buffer against self-targeting, since damage to genomic DNA can be efficiently repaired by utilizing intact genome copies as repair templates for homologous recombination. Furthermore, an ineffective autoimmune reaction seems to lead to the acquisition of spacers originating from genomic DNA. Finally, the application of the endogenous CRISPR-Cas system of H. volcanii as a tool for repression of gene expression was established. To achieve this, the interference complex was reprogrammed with synthetic crRNAs mediating the binding of a desired target sequence. That way, the expression of a plasmid-encoded gene, several genomic genes as well as an essential gene could be inhibited. Several attempts to optimize the system revealed that the expression of a catalytically inactive Cas3-variant could further enhance the efficiency of gene repression and a shortening of the spacer sequence by 5 nt was possible, however crRNA synthesis must occur by a mechanism that generates correct ends.dc.description.abstract
Verbreitende StelleUniversität Ulmdc.publisher
Link zum Lizenztexthttps://oparu.uni-ulm.de/xmlui/license_v3dc.rights.uri
DDC-SachgruppeDDC 570 / Life sciencesdc.subject.ddc
MeSHCRISP-Cas systemsdc.subject.mesh
MeSHHaloferax volcaniidc.subject.mesh
MeSHClustered regularly interspaced short palindromic repeatsdc.subject.mesh
TitelDas CRISPR-Cas-System von Haloferax volcanii: CRISPRi und Autoimmunitätdc.title
Datum der Annahme2017-07-13dcterms.dateAccepted
GutachterMarchfelder, Anitadc.contributor.referee
GutachterBinder, Stefandc.contributor.referee
GNDGenome Editingdc.subject.gnd
GNDHaloferax volcaniidc.subject.gnd
FakultätFakultät für Naturwissenschaftenuulm.affiliationGeneral
InstitutionInstitut für Molekulare Botanikuulm.affiliationSpecific
Titelverleihende FakultätFakultät für Naturwissenschaftenuulm.thesisGrantor

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