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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.
Pubmed ID: 27614072 RIS Download
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Software repository for R packages related to analysis and comprehension of high throughput genomic data. Uses separate set of commands for installation of packages. Software project based on R programming language that provides tools for analysis and comprehension of high throughput genomic data.
View all literature mentionsFunctional genomics data repository supporting MIAME-compliant data submissions. Includes microarray-based experiments measuring the abundance of mRNA, genomic DNA, and protein molecules, as well as non-array-based technologies such as serial analysis of gene expression (SAGE) and mass spectrometry proteomic technology. Array- and sequence-based data are accepted. Collection of curated gene expression DataSets, as well as original Series and Platform records. The database can be searched using keywords, organism, DataSet type and authors. DataSet records contain additional resources including cluster tools and differential expression queries.
View all literature mentionsSoftware package for the analysis of gene expression microarray data, especially the use of linear models for analyzing designed experiments and the assessment of differential expression.
View all literature mentionsFunctional genomics data repository supporting MIAME-compliant data submissions. Includes microarray-based experiments measuring the abundance of mRNA, genomic DNA, and protein molecules, as well as non-array-based technologies such as serial analysis of gene expression (SAGE) and mass spectrometry proteomic technology. Array- and sequence-based data are accepted. Collection of curated gene expression DataSets, as well as original Series and Platform records. The database can be searched using keywords, organism, DataSet type and authors. DataSet records contain additional resources including cluster tools and differential expression queries.
View all literature mentionsCell line HEK293 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsCell line Huh-7 is a Cancer cell line with a species of origin Homo sapiens (Human)
View all literature mentionsCell line Huh-7.5 is a Cancer cell line with a species of origin Homo sapiens (Human)
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