Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.

IF 2.3 2区 农林科学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Jacob N Dayton,Tammy T Tran,Elisa Saint-Denis,Erik B Dopman
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Abstract

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.
欧洲玉米螟(Ostrinia nubilalis)中高效的 CRISPR/Cas9 介导的基因组编辑。
欧洲玉米螟(Ostrinia nubilalis)是一种农业害虫,也是研究物种、季节适应性和昆虫抗性管理的新兴模式。尽管以前的工作已经发现了与欧洲玉米螟生命周期、繁殖和对 Bt 毒素的抗性差异有关的基因,但由于普遍缺乏针对欧洲玉米螟的强大的基因编辑方案,候选基因的功能验证一直是个障碍。在这里,我们利用 CRISPR/Cas9 基因组编辑系统展示了一种高效实用的方法来诱变 O. nubilalis 的可遗传基因。我们在两个昼夜节律时钟基因--周期(per)和色素分散因子受体(pdfr)以及一个发育基因--前胸托叶激素(ptth)上产生了精确的功能缺失(LOF)突变。由于不需要可见的遗传标记,不同的单导RNA(sgRNA)的基因编辑效率仍然很高,突变对F1后代的种系传递接近100%。当注射高浓度的单导RNA或双导RNA时,F0突变体在行为和发育方面会出现基因特异性表型差异。具体而言,F0 基因突变体表明,PER(而非 PDFR)对正常的蜕皮时间至关重要。PTTH F0突变体明显更重,并表现出更高的休眠率。这项工作将加速未来对幼虫基因功能的研究,并促进在其他鳞翅目昆虫和非模式昆虫中开展类似的筛选工作。
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来源期刊
Insect Molecular Biology
Insect Molecular Biology 生物-昆虫学
CiteScore
4.80
自引率
3.80%
发文量
68
审稿时长
6-12 weeks
期刊介绍: Insect Molecular Biology has been dedicated to providing researchers with the opportunity to publish high quality original research on topics broadly related to insect molecular biology since 1992. IMB is particularly interested in publishing research in insect genomics/genes and proteomics/proteins. This includes research related to: • insect gene structure • control of gene expression • localisation and function/activity of proteins • interactions of proteins and ligands/substrates • effect of mutations on gene/protein function • evolution of insect genes/genomes, especially where principles relevant to insects in general are established • molecular population genetics where data are used to identify genes (or regions of genomes) involved in specific adaptations • gene mapping using molecular tools • molecular interactions of insects with microorganisms including Wolbachia, symbionts and viruses or other pathogens transmitted by insects Papers can include large data sets e.g.from micro-array or proteomic experiments or analyses of genome sequences done in silico (subject to the data being placed in the context of hypothesis testing).
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