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TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
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| Veröffentlicht in: | Nucleic acids research 45(2017,1) Artikel-Nummer e3, 17 Seiten |
|---|---|
| Personen und Körperschaften: | , , , , , , , , , |
| Titel: | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus/ 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 |
| Format: | E-Book-Kapitel |
| Sprache: | Englisch |
| veröffentlicht: |
2017
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| Gesamtaufnahme: |
: Nucleic acids research, 45(2017,1) Artikel-Nummer e3, 17 Seiten
, volume:45 |
| Quelle: | Verbunddaten SWB Lizenzfreie Online-Ressourcen |
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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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| author | Senís Herrero, Elena, Mockenhaupt, Stefan, Rupp, Daniel, Paramasivam, Nagarajan, Große, Stefanie, Windisch, Marc Peter, Schmidt, Florian, Eils, Roland, Bartenschlager, Ralf, Grimm, Dirk |
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| contents | 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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| spelling | Senís Herrero, Elena VerfasserIn (DE-588)1080250026 (DE-627)844088617 (DE-576)453431690 aut, TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus 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, 2017, 17, Text txt rdacontent, Computermedien c rdamedia, Online-Ressource cr rdacarrier, Published online: 9 September 2016, Gesehen am 06.07.2018, 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., 2016, Mockenhaupt, Stefan VerfasserIn (DE-588)102293662X (DE-627)717335593 (DE-576)366194259 aut, Rupp, Daniel 1984- VerfasserIn (DE-588)1023581426 (DE-627)718413725 (DE-576)367031906 aut, Paramasivam, Nagarajan 1985- VerfasserIn (DE-588)1027462979 (DE-627)729165485 (DE-576)373239238 aut, Große, Stefanie VerfasserIn (DE-588)1074036786 (DE-627)830179054 (DE-576)435541285 aut, Windisch, Marc Peter VerfasserIn (DE-588)133084051 (DE-627)534477585 (DE-576)299611868 aut, Schmidt, Florian VerfasserIn (DE-588)1021984884 (DE-627)715845276 (DE-576)364415436 aut, Eils, Roland 1965- VerfasserIn (DE-588)1020648287 (DE-627)691291705 (DE-576)361718195 aut, Bartenschlager, Ralf 1958- VerfasserIn (DE-588)1058097989 (DE-627)796390509 (DE-576)168706067 aut, Grimm, Dirk VerfasserIn (DE-588)1016476671 (DE-627)70553376X (DE-576)352260645 aut, Enthalten in Nucleic acids research Oxford : Oxford Univ. Press, 1974 45(2017,1) Artikel-Nummer e3, 17 Seiten Online-Ressource (DE-627)26813250X (DE-600)1472175-2 (DE-576)07760878X 1362-4962 nnns, volume:45 year:2017 number:1 extent:17, http://dx.doi.org/10.1093/nar/gkw805 Verlag Resolving-System kostenfrei Volltext, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224498/ Verlag kostenfrei Volltext, http://dx.doi.org/10.1093/nar/gkw805 LFER, LFER 2018-07-10T00:00:00Z |
| spellingShingle | Senís Herrero, Elena, Mockenhaupt, Stefan, Rupp, Daniel, Paramasivam, Nagarajan, Große, Stefanie, Windisch, Marc Peter, Schmidt, Florian, Eils, Roland, Bartenschlager, Ralf, Grimm, Dirk, 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) 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. |
| swb_id_str | 507382889 |
| title | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus |
| title_auth | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus |
| title_full | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus 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 |
| title_fullStr | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus 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 |
| title_full_unstemmed | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus 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 |
| title_in_hierarchy | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus / 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, |
| title_short | TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus |
| title_sort | talen crispr mediated engineering of a promoterless anti viral rnai hairpin into an endogenous mirna locus |
| url | http://dx.doi.org/10.1093/nar/gkw805, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224498/ |