Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases
- 자연과학대학 화학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 화학부(생화학전공), 2014. 2. 김진수.
- Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) , engineered nucleases, are composed of designable DNA-binding domains and a non-specific nuclease domain and enable a broad range of genomic modification by inducing double-strand breaks (DSBs) that stimulate intrinsic cellular repair mechanisms such as non-homologous recombination (NHEJ) and homologous recombination (HR) at specific genomic locations. This technology has been described earlier as promising tools for targeted genome engineering in cells and many organisms. Recently, RNA-guided endonucleases (RGENs) derived from bacterial type-II CRISPR/Cas system, have been described as site-specific endonucleases whose specificities are programmed by small RNA components. RGEN also has been applied in cells and organisms as genome engineering tool. Here in this study, injection of RGENs as Cas9 protein: guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell stage embryos of mice and zebrafish efficiently disrupts a target gene in both species. RGENs efficiently generated germ-line 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. I propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically-engineered model organisms accelerating functional genomic research.