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Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

Author

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  • Takahito Watanabe

    (Institute of Technology and Science, University of Tokushima)

  • Hiroshi Ochiai

    (Graduate School of Science, Hiroshima University)

  • Tetsushi Sakuma

    (Graduate School of Science, Hiroshima University)

  • Hadley W. Horch

    (Bowdoin College)

  • Naoya Hamaguchi

    (Institute of Technology and Science, University of Tokushima)

  • Taro Nakamura

    (Institute of Technology and Science, University of Tokushima)

  • Tetsuya Bando

    (Institute of Technology and Science, University of Tokushima)

  • Hideyo Ohuchi

    (Institute of Technology and Science, University of Tokushima)

  • Takashi Yamamoto

    (Graduate School of Science, Hiroshima University)

  • Sumihare Noji

    (Institute of Technology and Science, University of Tokushima)

  • Taro Mito

    (Institute of Technology and Science, University of Tokushima)

Abstract

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

Suggested Citation

  • Takahito Watanabe & Hiroshi Ochiai & Tetsushi Sakuma & Hadley W. Horch & Naoya Hamaguchi & Taro Nakamura & Tetsuya Bando & Hideyo Ohuchi & Takashi Yamamoto & Sumihare Noji & Taro Mito, 2012. "Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases," Nature Communications, Nature, vol. 3(1), pages 1-8, January.
    • Handle: RePEc:nat:natcom:v:3:y:2012:i:1:d:10.1038_ncomms2020
    DOI: 10.1038/ncomms2020
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    Cited by:

    1. Victor Zeng & Ben Ewen-Campen & Hadley W Horch & Siegfried Roth & Taro Mito & Cassandra G Extavour, 2013. "Developmental Gene Discovery in a Hemimetabolous Insect: De Novo Assembly and Annotation of a Transcriptome for the Cricket Gryllus bimaculatus," PLOS ONE, Public Library of Science, vol. 8(5), pages 1-22, May.

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