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Nucleic Acids Res. 2017 Jan 9;45(1):e3. doi: 10.1093/nar/gkw805. Epub 2016 Sep 9.

TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus.

Author information

1
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.
2
BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
3
Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, 69120, Germany.
4
Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
5
Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
6
Medical Faculty Heidelberg, Heidelberg University, Heidelberg, 69120, Germany.
7
Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, 85764, Germany.
8
Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, 69120, Germany.
9
Department of Mathematics, Technische Universität München, Garching, 85748, Germany.
10
Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, 69120, Germany.
11
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany dirk.grimm@bioquant.uni-heidelberg.de.

Abstract

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.

PMID:
27614072
PMCID:
PMC5224498
DOI:
10.1093/nar/gkw805
[Indexed for MEDLINE]
Free PMC Article

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