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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas systems provide targeted genome modification capabilities.
• RNA-guided endonucleases (RGENs) are derived from prokaryotic Type II CRISPR-Cas systems.

Purpose of the Study:

• To establish gene-knockout mice and zebrafish using RGENs.
• To assess the efficiency and toxicity of RGEN-mediated mutagenesis in animal models.

Main Methods:

• Injection of Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of mice and zebrafish.
• Analysis of germline transmittable mutations and phenotypic effects.

Main Results:

• RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice.
• Minimal toxicity was observed with RGEN application.
• RGEN-induced mutations in the mouse Prkdc gene led to immunodeficiency in F₀ and F₁ generations.

Conclusions:

• RGEN-mediated mutagenesis is an efficient method for creating gene-knockout animals.
• This technology significantly accelerates the generation of genetically engineered model organisms.
• RGENs offer a promising tool for advancing functional genomic research.