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

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

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.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Disease Resistance / genetics
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Editing
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Genetic Engineering*
  • Genetic Loci
  • Genome, Human
  • HEK293 Cells
  • Hepacivirus / genetics*
  • Hepatocytes / metabolism
  • Hepatocytes / virology
  • Host-Pathogen Interactions
  • Humans
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • RNA Interference*
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • Sequence Analysis, DNA
  • Transcription Activator-Like Effector Nucleases / genetics*
  • Transcription Activator-Like Effector Nucleases / metabolism
  • Virus Replication / genetics

Substances

  • Bacterial Proteins
  • MIRN122 microRNA, human
  • MicroRNAs
  • RNA, Small Interfering
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • Transcription Activator-Like Effector Nucleases