Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.
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.
Doyon
Yannick
Y
Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, California 94804, USA.
McCammon
Jasmine M
JM
Miller
Jeffrey C
JC
Faraji
Farhoud
F
Ngo
Catherine
C
Katibah
George E
GE
Amora
Rainier
R
Hocking
Toby D
TD
Zhang
Lei
L
Rebar
Edward J
EJ
Gregory
Philip D
PD
Urnov
Fyodor D
FD
Amacher
Sharon L
SL
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*
D029961:Zebrafish Proteins; D003851:Deoxyribonucleases