Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome
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.
Key wordsZebrafish CRISPR/Cas Targeted transgene integration Genome modification Genome editing Genome engineering Site-specific nuclease Homology-independent repair
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.
- 13.Balciuniene J, Balciunas D (2013) Gene trapping using gal4 in zebrafish. J Vis Exp (79):e50113Google Scholar
- 53.Westerfield M (ed) (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edn. University of Orgeon Press, OregonGoogle Scholar
- 59.Sambrook J, Russell DW (2006) Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc 2006Google Scholar