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

作者：Watanabe Takahito; Ochiai Hiroshi; Sakuma Tetsushi; Horch Hadley W; Hamaguchi Naoya; Nakamura Taro; Bando Tetsuya; Ohuchi Hideyo; Yamamoto Takashi; Noji Sumihare; Mito Taro^*

来源：Nature Communications, 2012, 3: 1017.

DOI：10.1038/ncomms2020

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.

• 出版日期2012-8