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

Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Hadley W. Horch, Naoya Hamaguchi, Taro Nakamura, Tetsuya Bando, Hideyo Ohuchi, Takashi Yamamoto, Sumihare Noji and Taro Mito ()
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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

Nature Communications, 2012, vol. 3, issue 1, 1-8

Abstract: 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.

Date: 2012
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DOI: 10.1038/ncomms2020

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