Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases

Genome Res. 2014 Jan;24(1):125-31. doi: 10.1101/gr.163394.113. Epub 2013 Nov 19.


RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F₀ and F₁ mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn / genetics
  • CRISPR-Associated Proteins / metabolism*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Embryo, Mammalian
  • Embryo, Nonmammalian
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Forkhead Transcription Factors / metabolism
  • Gene Knockout Techniques
  • Genome
  • Germ-Line Mutation
  • Mice
  • Mice, Inbred BALB C
  • Mice, Knockout
  • Mutagenesis*
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism
  • Phenotype
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Zebrafish / genetics
  • Zebrafish / metabolism


  • CRISPR-Associated Proteins
  • Forkhead Transcription Factors
  • Nuclear Proteins
  • RNA, Guide
  • Endonucleases