Designer nuclease-mediated gene correction via homology-directed repair in an in vitro model of canine hemophilia B
| Autor: | Thorsten Bergmann, Verena Schildgen, Eric Ehrke-Schulz, Jian Gao, Oliver Schildgen, Anja Ehrhardt, Maren Schiwon, Stephan David |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Locus (genetics) Computational biology Biology Hemophilia B Homology directed repair Factor IX 03 medical and health sciences Dogs Drug Discovery Genetics CRISPR Animals Humans DNA Breaks Double-Stranded Dog Diseases Molecular Biology Gene Genetics (clinical) Gene Editing Nuclease Cas9 Point mutation Recombinational DNA Repair Endonucleases genomic DNA 030104 developmental biology HEK293 Cells Gene Targeting biology.protein Molecular Medicine CRISPR-Cas Systems Genetic Engineering |
| Zdroj: | The journal of gene medicine. 20(5) |
| ISSN: | 1521-2254 |
| Popis: | BACKGROUND Gene correction at specific target loci provides a powerful strategy for overcoming genetic diseases. In the present study, we aimed to use an in vitro model for canine hemophilia B containing a single point mutation in the catalytic domain of the canine coagulation factor IX (cFIX) gene. To correct the defective gene via homology-directed repair (HDR), we designed transcription-activator like effector nucleases and clustered regularly interspaced short palindromic repeats including Cas9 (CRISPR/Cas9) for introduction of double-strand breaks at the mutation site. METHODS To generate a stable cell line containing the mutated cFIX locus, a 2-kb genomic DNA fragment derived from a hemophilia B dog was amplified and integrated utilizing the phiC31 integrase system. Designer nucleases were assembled and cloned into vectors for constitutive and inducible expression. To detect mutations, insertions and deletions, and HDR events after nuclease treatment T7E1 assays, an amplification-refractory mutation system-quantitative polymerase chain reaction and pyrosequencing were performed. RESULTS To perform HDR correction experiments, we established a cell line carrying the mutated cFIX locus. In HDR approaches we either explored a wild-type or an optimized cFIX sequence and we found that our modified HDR cassette showed higher gene correction efficiencies of up to 6.4%. Furthermore, we compared inducible and constitutive designer nuclease expression systems and found that the inducible system resulted in comparable HDR efficiencies. CONCLUSIONS In conclusion, the present study demonstrates the potential of this strategy for gene therapeutic approaches in vitro and in a canine model for hemophilia B. |
| Databáze: | OpenAIRE |
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