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Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome

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Zebrafish

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1451))

Abstract

Targeting nucleases like zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system have revolutionized genome-editing possibilities in many model organisms. They allow the generation of loss-of-function alleles by the introduction of double-strand breaks at defined sites within genes, but also more sophisticated genome-editing approaches have become possible. These include the integration of donor plasmid DNA into the genome by homology-independent repair mechanisms after CRISPR/Cas9-mediated cleavage. Here we present a protocol outlining the most important steps to target a genomic site and to integrate a donor plasmid at this defined locus.

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Acknowledgments

A special thanks to J. P. Concordet, K. Duroure, and A. De Cian for helping with the development and initial establishment of the homology-independent targeting strategy. We would like to thank J. Wittbrodt for scientific discussion and support and members of the Del Bene lab for general discussion and comments. The Del Bene lab “Neural Circuits Development” is part of the Laboratoire d’Excellence (LabEx) entitled DEEP (ANR-11-LABX-0044) and the Ecole des Neurosciences de Paris. T.O.A. was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship. This work has been supported by ATIP/AVENIR program starting grant (FDB), ERC-StG #311159 (FDB), CNRS, INSERM, and Institut Curie.

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Authors and Affiliations

  1. Neuronal Circuit Development Group, Unité de Génétique et Biologie du Développement, U934/UMR3215, Pole de Biologie du Développement et Cancer, Institut Curie—Centre de Recherche, 26, rue d’Ulm, 75248, Paris Cedex 05, France

    Thomas O. Auer Ph.D. & Filippo Del Bene Ph.D.

  2. CNRS UMR 3215, 75248, Paris, France

    Thomas O. Auer Ph.D. & Filippo Del Bene Ph.D.

  3. INSERM U934, 75248, Paris, France

    Thomas O. Auer Ph.D. & Filippo Del Bene Ph.D.

  4. Centre for Organismal Studies Heidelberg, University of Heidelberg, Im Neuenheimer Feld 230, 69120, Heidelberg, Germany

    Thomas O. Auer Ph.D.

  5. Center for Integrative Genomics, University of Lausanne, Génopode Building, CH-1015, Lausanne, Switzerland

    Thomas O. Auer Ph.D.

Authors
  1. Thomas O. Auer Ph.D.
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  2. Filippo Del Bene Ph.D.
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Corresponding authors

Correspondence to Thomas O. Auer Ph.D. or Filippo Del Bene Ph.D. .

Editor information

Editors and Affiliations

  1. Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Japan

    Koichi Kawakami

  2. Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit & The University of Edinburgh Cancer Research UK Centre, Edinburgh, United Kingdom

    E. Elizabeth Patton

  3. Champalimaud Centre for the Unknown, Champalimaud Neuroscience Programme Champalimaud Centre for the Unknown, Lisbon, Portugal

    Michael Orger

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Auer, T.O., Del Bene, F. (2016). Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome. In: Kawakami, K., Patton, E., Orger, M. (eds) Zebrafish. Methods in Molecular Biology, vol 1451. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3771-4_3

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  • DOI: https://doi.org/10.1007/978-1-4939-3771-4_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3769-1

  • Online ISBN: 978-1-4939-3771-4

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