Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair

  1. Filippo Del Bene1,2,3,8
  1. 1Institut Curie, Centre de Recherche, Paris F-75248, France;
  2. 2CNRS UMR 3215, Paris F-75248, France;
  3. 3INSERM U934, F-75248 Paris, France;
  4. 4Centre for Organismal Studies Heidelberg, University of Heidelberg, 69120 Heidelberg, Germany;
  5. 5Muséum National d'Histoire Naturelle, Paris F-75231, France;
  6. 6CNRS UMR 7196, Paris F-75231, France;
  7. 7INSERM U565, Paris F-75231, France

    Abstract

    Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

    Footnotes

    • 8 Corresponding authors

      E-mail filippo.del-bene{at}curie.fr

      E-mail jean-paul.concordet{at}mnhn.fr

    • [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.161638.113.

    • Received June 5, 2013.
    • Accepted October 28, 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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