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

• DOI: 10.1038/ncomms2020
• Publication Date: 2012-08-21T09:50:30Z
• Authors: Takahito Watanabe; Hiroshi Ochiai; Tetsushi Sakuma; Hadley W. Horch; Naoya Hamaguchi; Taro Nakamura; Tetsuya Bando; Hideyo Ohuchi; Takashi Yamamoto; Sumihare Noji; Taro Mito
• Keywords: Zinc finger nuclease; Genome Engineering; Gene targeting; Nuclease; Gene knockout

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