TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) s...
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| Main Authors: | , , , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
2017
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| In: |
Nucleic acids research
Year: 2016, Volume: 45, Issue: 1 |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/gkw805 |
| Online Access: | Verlag, kostenfrei, Volltext: http://dx.doi.org/10.1093/nar/gkw805 Verlag, kostenfrei, Volltext: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224498/ 
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| Author Notes: | Elena Senís, Stefan Mockenhaupt, Daniel Rupp, Tobias Bauer, Nagarajan Paramasivam, Bettina Knapp, Jan Gronych, Stefanie Grosse, Marc P. Windisch, Florian Schmidt, Fabian J. Theis, Roland Eils, Peter Lichter, Matthias Schlesner, Ralf Bartenschlager and Dirk Grimm |
MARC
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| 520 | |a Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy. | ||
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