Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

中文翻译：

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在精确的单一碱基置换shibire通过CRISPR / Cas9介导的同源性定向修复基因昆士兰实蝇

使用不育昆虫技术（SIT）消灭有害生物涉及高密度释放的雄性雄性，与野生雌性交配并最终抑制了种群。不需要对雌性进行SIT消毒，并且在释放前将其从雄性中移除或分离仍然具有挑战性。为了开发遗传性菌株（GSS），需要条件性状，例如对温度敏感的杀伤力。在这里，我们将已知的果蝇黑腹果蝇对温度敏感的胚胎致死突变引入到Bactrocera tryoni（澳大利亚的一种严重园艺害虫）中。D. melanogaster基因shibire中的一个非同义点突变会在29°C下导致胚胎致死率，我们成功地使用CRISPR / Cas9技术在Tryoni中重建了直系同源的Shibire温度敏感1（shits1）突变。经过三代人的基因型分析显示，高适应性成本与shits1突变体等位基因相关，并且shits1纯合子在21°C下不可行，这比黑腹果蝇中记录的表型更为严重。我们已经证明了CRISPR / Cas9首次成功用于通过农业害虫昆虫中的同源性定向修复在内源基因中引入精确的单碱基替代，并且该技术可以用于试验其他条件突变，最终产生遗传性别SIT的菌株。