Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees
Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees
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DOI：
10.1080/01677063.2021.1887173
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发表时间：
2021-02-08
影响因子：
1.9
通讯作者：
Robinson, Gene E.
中科院分区：
文献类型：
--
作者：
Chen, Zhenqing;Traniello, Ian M.;Robinson, Gene E.
在昆虫中，气味受体促进嗅觉交流，并需要高度保守的辅助受体基因orco的功能。对几种蚂蚁和飞蛾的基因组编辑研究表明，除了在成年嗅觉中的典型作用外，orco还可以具有神经发育功能，这一作用首先在黑腹果蝇中发现。为了扩展这一分析，我们确定了orco突变是否也影响蜜蜂（Apis mellifera）成年大脑的发育，蜜蜂是社会行为和化学通讯的重要模型系统。我们使用CRISPR/Cas9敲除orco，并检查了解剖和分子结果。为了提高效率，我们将胚胎显微注射与实验室卵子收集和体外饲养系统相结合。这种新的工作流程通过克服与实地研究相关的限制，推进了蜜蜂的基因组工程技术。我们使用桑格测序来快速选择具有完全orco敲除的个体用于神经解剖学分析，随后使用扩增子测序来验证和描述突变。与野生型对照相比，突变蜜蜂的触角叶中肾小球显著减少，肾小球总体积较小，平均单个肾小球体积较高。RNA测序显示，orco敲除还导致触角中数百个基因的差异表达，包括与神经发育相关的基因和编码气味受体的基因。其他类型的化学感受器基因的表达通常不受影响，反映了本研究中CRISPR活性的特异性。这些结果表明，orco的神经发育效应与特定的昆虫生活史。
In insects, odorant receptors facilitate olfactory communication and require the functionality of the highly conserved co-receptor gene orco. Genome editing studies in a few species of ants and moths have revealed that orco can also have a neurodevelopmental function, in addition to its canonical role in adult olfaction, discovered first in Drosophila melanogaster. To extend this analysis, we determined whether orco mutations also affect the development of the adult brain of the honey bee Apis mellifera, an important model system for social behavior and chemical communication. We used CRISPR/Cas9 to knock out orco and examined anatomical and molecular consequences. To increase efficiency, we coupled embryo microinjection with a laboratory egg collection and in vitro rearing system. This new workflow advances genomic engineering technologies in honey bees by overcoming restrictions associated with field studies. We used Sanger sequencing to quickly select individuals with complete orco knockout for neuroanatomical analyses and later validated and described the mutations with amplicon sequencing. Mutant bees had significantly fewer glomeruli, smaller total volume of all the glomeruli, and higher mean individual glomerulus volume in the antennal lobe compared to wild-type controls. RNA-Sequencing revealed that orco knockout also caused differential expression of hundreds of genes in the antenna, including genes related to neural development and genes encoding odorant receptors. The expression of other types of chemoreceptor genes was generally unaffected, reflecting specificity of CRISPR activity in this study. These results suggest that neurodevelopmental effects of orco are related to specific insect life histories.