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TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus.
Senís, Elena; Mockenhaupt, Stefan; Rupp, Daniel; Bauer, Tobias; Paramasivam, Nagarajan; Knapp, Bettina; Gronych, Jan; Grosse, Stefanie; Windisch, Marc P; Schmidt, Florian; Theis, Fabian J; Eils, Roland; Lichter, Peter; Schlesner, Matthias; Bartenschlager, Ralf; Grimm, Dirk.
Afiliación
  • Senís E; Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.
  • Mockenhaupt S; BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
  • Rupp D; Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.
  • Bauer T; BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
  • Paramasivam N; Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, 69120, Germany.
  • Knapp B; Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
  • Gronych J; Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
  • Grosse S; Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
  • Windisch MP; Medical Faculty Heidelberg, Heidelberg University, Heidelberg, 69120, Germany.
  • Schmidt F; Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, 85764, Germany.
  • Theis FJ; Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, 69120, Germany.
  • Eils R; Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.
  • Lichter P; BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
  • Schlesner M; Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, 69120, Germany.
  • Bartenschlager R; Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.
  • Grimm D; BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
Nucleic Acids Res ; 45(1): e3, 2017 01 09.
Article en En | MEDLINE | ID: mdl-27614072
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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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ingeniería Genética / Hepacivirus / MicroARNs / ARN Interferente Pequeño / Interferencia de ARN / Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas / Nucleasas de los Efectores Tipo Activadores de la Transcripción Límite: Humans Idioma: En Revista: Nucleic Acids Res Año: 2017 Tipo del documento: Article País de afiliación: Alemania Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ingeniería Genética / Hepacivirus / MicroARNs / ARN Interferente Pequeño / Interferencia de ARN / Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas / Nucleasas de los Efectores Tipo Activadores de la Transcripción Límite: Humans Idioma: En Revista: Nucleic Acids Res Año: 2017 Tipo del documento: Article País de afiliación: Alemania Pais de publicación: Reino Unido