title: Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome.
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.
authors.affiliation: 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.auer@unil.ch.|CNRS UMR 3215, 75248, Paris, France. thomas.auer@unil.ch.|INSERM U934, 75248, Paris, France. thomas.auer@unil.ch.|Centre for Organismal Studies Heidelberg, University of Heidelberg, Im Neuenheimer Feld 230, 69120, Heidelberg, Germany. thomas.auer@unil.ch.|Center for Integrative Genomics, University of Lausanne, Génopode Building, CH-1015, Lausanne, Switzerland. thomas.auer@unil.ch.
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Auer
authors.affiliation: 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. filippo.del-bene@curie.fr.|CNRS UMR 3215, 75248, Paris, France. filippo.del-bene@curie.fr.|INSERM U934, 75248, Paris, France. filippo.del-bene@curie.fr.
authors.forename: Filippo
authors.initials: F
authors.lastname: Del Bene
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D005818:Genetic Engineering; D010957:Plasmids / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics*
keywords: CRISPR/Cas; Genome editing; Genome engineering; Genome modification; Homology-independent repair; Site-specific nuclease; Targeted transgene integration; Zebrafish