CRISPR/Cas9-mediated genome editing of splicing mutation causing congenital hearing loss
| Autor: | Nari Ryu, Kyu-Yup Lee, Min-A Kim, Jong Kyung Sonn, Ye-Ri Kim, Deok-Gyun Choi, Un-Kyung Kim |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Silent mutation Staphylococcus aureus Biology Cell Line Mice 03 medical and health sciences Exon 0302 clinical medicine Bacterial Proteins Genome editing CRISPR-Associated Protein 9 otorhinolaryngologic diseases Genetics Animals Humans CRISPR Hearing Loss Gene Editing Cas9 Intron Gene targeting Exons Genetic Therapy General Medicine 030104 developmental biology Sulfate Transporters 030220 oncology & carcinogenesis Gene Targeting RNA splicing CRISPR-Cas Systems |
| Zdroj: | Gene. 703:83-90 |
| ISSN: | 0378-1119 |
| Popis: | Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss. |
| Databáze: | OpenAIRE |
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