Engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE68638 | GEO | 2015/05/07
SECONDARY ACCESSION(S): PRJNA283273
REPOSITORIES: GEO
Similar Datasets
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-05-07 | E-GEOD-68638 | biostudies-arrayexpress
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. 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 RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy Four Huh7 cells lines at 3 different passages were analyzed. The reference cell line was Huh7 wild type cells (WT). The other three cell lines had an integration of an anti-HCV shmiRNA in the hcr locus and miR-122 intact (T2 31.3) or mutated (TS 30.20 and U6 20.16). RNA was extracted from three different passages.
2015-05-07 | E-GEOD-68620 | biostudies-arrayexpress
Project description: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.
| S-EPMC5224498 | biostudies-literature
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. 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 RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy
2015-05-07 | GSE68620 | GEO
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. 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 RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy Four Huh7 cells lines at 3 different passages were analyzed. The reference cell line was Huh7 wild type cells (WT). The other three cell lines had an integration of an anti-HCV shmiRNA in the hcr locus and miR-122 intact (T2 31.3) or mutated (TS 30.20 and U6 20.16). RNA was extracted from three different passages.
2015-05-07 | E-GEOD-68637 | biostudies-arrayexpress
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. 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 should benefit numerous applications, from basic miRNA research, to human cell and gene therapy.
| EGAS00001001252 | EGA
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. 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 RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy
2015-05-07 | GSE68637 | GEO
Project description:Engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus
| PRJNA283273 | ENA
Project description:This SuperSeries is composed of the SubSeries listed below.
2020-10-30 | GSE160425 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-06-01 | E-GEOD-41058 | biostudies-arrayexpress