title: Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.
abstract: We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.
authors.affiliation: Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, California 94804, USA.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Doyon
authors.forename: Jasmine M
authors.initials: JM
authors.lastname: McCammon
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Farhoud
authors.initials: F
authors.lastname: Faraji
authors.forename: Catherine
authors.initials: C
authors.lastname: Ngo
authors.forename: George E
authors.initials: GE
authors.lastname: Katibah
authors.forename: Rainier
authors.initials: R
authors.lastname: Amora
authors.forename: Toby D
authors.initials: TD
authors.lastname: Hocking
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Amacher
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000502:physiology*; D003851:Deoxyribonucleases / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D029961:Zebrafish Proteins; D003851:Deoxyribonucleases