title: Precise Editing of the Zebrafish Genome Made Simple and Efficient.
abstract: We present simple and efficient methods for creating heritable modifications of the zebrafish genome. Precisely modified alleles are generated by homologous recombination between the host genome and dsDNA donor molecules, stimulated by the induction of chromosomally targeted double-strand breaks. Several kilobase-long tracts of genome sequence can be replaced. Tagging donor sequences with reporter genes that can be subsequently excised improves recovery of edited alleles by an order of magnitude and facilitates recovery of recessive and phenotypically silent conditional mutations. We generate and demonstrate functionality of (1) alleles with a single codon change, (2) an allele encoding an epitope-tagged version of an endogenous protein, (3) alleles expressing reporter proteins, and (4) a conditional allele in which an exon is flanked by recombinogenic loxP sites. Our methods make recovery of a broad range of genome editing events very practicable, significantly advancing applicability of the zebrafish for studying normal biological processes and modeling diseases.
authors.affiliation: Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Hoshijima
authors.affiliation: Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Jurynec
authors.affiliation: Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA. Electronic address: grunwald@genetics.utah.edu.
authors.forename: David Jonah
authors.initials: DJ
authors.lastname: Grunwald
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D059765:Homologous Recombination; D023421:Models, Animal; D008957:Models, Genetic; D017393:RNA Editing; D015027:Zebrafish / Q000002:abnormalities / Q000196:embryology / Q000235:genetics*