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

TLDR

RNA‑guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR‑Cas system, enable targeted genome modification in cells and organisms. The study establishes gene‑knockout mice and zebrafish by injecting Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one‑cell‑stage embryos, proposing that this approach will accelerate creation of genetically engineered model organisms. The authors generated knockouts by injecting Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one‑cell‑stage embryos of mice and zebrafish. RGENs produced germline‑transmittable mutations in up to 93 % of newborn mice with minimal toxicity, and targeting the Prkdc gene caused immunodeficiency in both F₀ and F₁ mice.

Abstract

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.