Young Hoon Sung1,5, Jong Min Kim2,5, Hyun-Taek Kim3,5, Jaehoon Lee1, Jisun Jeon1, Young Jin1, Jung-Hwa Choi3, Young Ho Ban1, Sang-Jun Ha1, Cheol-Hee Kim3, Han-Woong Lee1,4,6 and Jin-Soo Kim2,6
1Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea;
2National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea;
3Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea;
4Yonsei Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Republic of Korea
5 These authors contributed equally to this work
6 Corresponding authors
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.