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

  1. Jin-Soo Kim2,6
  1. 1Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea;
  2. 2National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Seoul 151-747, Republic of Korea;
  3. 3Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea;
  4. 4Yonsei Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Republic of Korea

    1. ↵5 These authors contributed equally to this work.

    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.

    Footnotes

    • ↵6 Corresponding authors

      E-mail jskim01{at}snu.ac.kr

      E-mail hwl{at}yonsei.ac.kr

    • [Supplemental material is available for this article.]

    • Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.163394.113.

    • Received July 11, 2013.
    • Accepted October 7, 2013.

    This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as described at http://creativecommons.org/licenses/by-nc/3.0/.

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    1. Published in Advance November 19, 2013, doi: 10.1101/gr.163394.113 Genome Res. 24: 125-131 © 2014 Sung et al.; Published by Cold Spring Harbor Laboratory Press

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