The Cre/loxP system belongs to the family of site-specific recombinases (SSR) that can precisely modify DNA that is flanked by two target sites. The reaction outcome is dependent on the structure and orientation of the target sites and includes excision, inversion and exchange of a DNA fragment. The system is established for more than 30 years and is active in vitro and in vivo in several organisms. These characteristics make the Cre/loxP system the ideal tool for genome editing. However, the strict target site preference for loxP limits its use to basic research where the loxP target sites can be introduced beforehand at the anticipated genomic locus. Directed evolution strategies have overcome this limitation and allow to generate Cre-like recombinases with altered DNA specificity. During this work, I developed the first dual-recombinase system based on evolved recombinases. Using two instead of one recombinase expands the targetability of the human genome by being more flexible in the target site search. After the identification of suitable target sites, I could show an evolved dual-recombinase system that can be used for excision and inversion of a human genomic locus. The recombinase mediated inversion reaction corrected a large genomic inversion that is frequently found in patients with severe Hemophilia A. Only two days after treating human cells with the developed dual-recombinase system, RecF8, 30% inversion could be detected in a human cell line. Applying RecF8 in patient specific endothelial cells corrected around 9% of the inversion back to the wild type sequence, which would be sufficient to drastically improve the quality of life of affected individuals. This genomic correction lead to the expression of the F8 gene, which is inactive elsewise. It remains to proof that the transcriptional reactivation of the F8 gene allows for the production of the Factor VIII protein. Before using RecF8 in a clinical setting, an in vivo study in a suitable mouse model is necessary. This study introduces a dual-recombinase system and thereby broadens the use of designer recombinases for genome editing. Moreover, in a proof on concept experiment this study shows that recombinases can be applied to correct large disease-causing genomic inversions in human cells. Altogether, the use of recombinases for scarless genome editing comes a step closer to reality.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:76927 |
| Date | 09 December 2021 |
| Creators | Lansing, Felix Johannes |
| Contributors | Buchholz, Frank, Guan, Kaomei, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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