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.
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
Elena
E
Senís
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
Stefan
S
Mockenhaupt
Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, 69120, Germany.|Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
Daniel
D
Rupp
Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
Tobias
T
Bauer
Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.|Medical Faculty Heidelberg, Heidelberg University, Heidelberg, 69120, Germany.
Nagarajan
N
Paramasivam
Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, 85764, Germany.
Bettina
B
Knapp
Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, 69120, Germany.
Jan
J
Gronych
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
Stefanie
S
Grosse
Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, 69120, Germany.
Marc P
MP
Windisch
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany.|BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
Florian
F
Schmidt
Institute of Computational Biology, Helmholtz Zentrum München, Neuherberg, 85764, Germany.|Department of Mathematics, Technische Universität München, Garching, 85748, Germany.
Fabian J
FJ
Theis
BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.|Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.|Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, 69120, Germany.
Roland
R
Eils
Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, 69120, Germany.
Peter
P
Lichter
Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
Matthias
M
Schlesner
Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, 69120, Germany.|Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany.
Ralf
R
Bartenschlager
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, Heidelberg, 69120, Germany dirk.grimm@bioquant.uni-heidelberg.de.|BioQuant Center, University of Heidelberg, Heidelberg, 69120, Germany.
Dirk
D
Grimm
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D060467:Disease Resistance / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D056426:Genetic Loci; D015894:Genome, Human; D057809:HEK293 Cells; D016174:Hepacivirus / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
D001426:Bacterial Proteins; C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D000069896:Transcription Activator-Like Effector Nucleases