CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

Sci Rep. 2015 Aug 5;5:12799. doi: 10.1038/srep12799.

Abstract

Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • CRISPR-Associated Protein 9
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA, Single-Stranded / genetics*
  • Endonucleases / genetics
  • Female
  • Gene Expression
  • Gene Knockdown Techniques / methods*
  • Genes, Reporter
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Introns
  • Male
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • MicroRNAs / genetics*
  • Microinjections
  • Mutagenesis, Insertional
  • Otx Transcription Factors / biosynthesis
  • Otx Transcription Factors / genetics
  • RNA Interference

Substances

  • Bacterial Proteins
  • DNA, Single-Stranded
  • MicroRNAs
  • Otx Transcription Factors
  • Otx2 protein, mouse
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • CRISPR-Associated Protein 9
  • Cas9 protein, Francisella novicida
  • Endonucleases