Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

Yannick Doyon; Jasmine M. McCammon; Jeffrey C. Miller; Farhoud Faraji; Catherine Ngo; George E. Katibah; Rainier Amora; Toby D. Hocking; Lei Zhang; Edward J. Rebar; Philip D. Gregory; Fyodor D. Urnov; Sharon L. Amacher

doi:10.1038/nbt1409

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

Yannick Doyon, Jasmine M. McCammon, Jeffrey C. Miller, Farhoud Faraji, Catherine Ngo, George E. Katibah, Rainier Amora, Toby D. Hocking, Lei Zhang, Edward J. Rebar, Philip D. Gregory, Fyodor D. Urnov, Sharon L. Amacher

Research output: Contribution to journal › Article › peer-review

772 Scopus citations

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.

Original language	English (US)
Pages (from-to)	702-708
Number of pages	7
Journal	Nature Biotechnology
Volume	26
Issue number	6
DOIs	https://doi.org/10.1038/nbt1409
State	Published - Jun 2008
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

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10.1038/nbt1409

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@article{95a71499b66f45c9862ba0696f46a695,

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.",

author = "Yannick Doyon and McCammon, {Jasmine M.} and Miller, {Jeffrey C.} and Farhoud Faraji and Catherine Ngo and Katibah, {George E.} and Rainier Amora and Hocking, {Toby D.} and Lei Zhang and Rebar, {Edward J.} and Gregory, {Philip D.} and Urnov, {Fyodor D.} and Amacher, {Sharon L.}",

note = "Funding Information: We thank Jasper Rine, Michael Holmes, Jacques C{\^o}t{\'e} and Amine Nourani for helpful suggestions regarding yeast-based screening of ZFNs, Randy Morse and Amine Nourani for providing reagents, Keith Cheng for gol cDNA, Keith Cheng and Herwig Baier for gol stocks, and Jennifer St. Hilaire, Emily Janus and Kimberly Blum for excellent zebrafish care and technical support. We thank the anonymous referees for helpful suggestions on the manuscript. The ntlb487 allele was isolated in Eugene, Oregon, in a National Institutes of Health (NIH)-funded screen (HD22486); S.L.A. gratefully acknowledges Charles Kimmel and our many colleagues at the University of Oregon who participated in mutagenesis screening. This work was supported by grants to S.L.A. from the NIH (1-R01-GM061952) and March of Dimes Birth Defects Foundation (1FY05-118). J.M.M. is supported by a National Science Foundation Predoctoral Fellowship.",

year = "2008",

month = jun,

doi = "10.1038/nbt1409",

language = "English (US)",

volume = "26",

pages = "702--708",

journal = "Nature Biotechnology",

issn = "1087-0156",

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number = "6",

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TY - JOUR

T1 - Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

AU - Doyon, Yannick

AU - McCammon, Jasmine M.

AU - Miller, Jeffrey C.

AU - Faraji, Farhoud

AU - Ngo, Catherine

AU - Katibah, George E.

AU - Amora, Rainier

AU - Hocking, Toby D.

AU - Zhang, Lei

AU - Rebar, Edward J.

AU - Gregory, Philip D.

AU - Urnov, Fyodor D.

AU - Amacher, Sharon L.

N1 - Funding Information: We thank Jasper Rine, Michael Holmes, Jacques Côté and Amine Nourani for helpful suggestions regarding yeast-based screening of ZFNs, Randy Morse and Amine Nourani for providing reagents, Keith Cheng for gol cDNA, Keith Cheng and Herwig Baier for gol stocks, and Jennifer St. Hilaire, Emily Janus and Kimberly Blum for excellent zebrafish care and technical support. We thank the anonymous referees for helpful suggestions on the manuscript. The ntlb487 allele was isolated in Eugene, Oregon, in a National Institutes of Health (NIH)-funded screen (HD22486); S.L.A. gratefully acknowledges Charles Kimmel and our many colleagues at the University of Oregon who participated in mutagenesis screening. This work was supported by grants to S.L.A. from the NIH (1-R01-GM061952) and March of Dimes Birth Defects Foundation (1FY05-118). J.M.M. is supported by a National Science Foundation Predoctoral Fellowship.

PY - 2008/6

Y1 - 2008/6

N2 - 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.

AB - 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.

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U2 - 10.1038/nbt1409

DO - 10.1038/nbt1409

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EP - 708

JO - Nature Biotechnology

JF - Nature Biotechnology

IS - 6

ER -

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

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