Department of Biology, University of Florida.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine.
J Vis Exp. 2021 Feb 9(168). doi: 10.3791/61930.
The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.
社会性昆虫的独特特征，如社会行为和生殖分工，是由它们的遗传系统控制的。为了研究基因如何调控社会特征，我们通过在同步胚胎阶段将 CRISPR 复合物递送至年轻胚胎中，开发了突变蚂蚁。在这里，我们提供了一种在 Harpegnathos saltator 中进行 CRISPR 介导的诱变的方案，这是一种表现出显著表型可塑性的 Ponera 蚂蚁物种。H. saltator 蚂蚁很容易在实验室环境中饲养。使用自制的石英针收集 Cas9 蛋白和体外合成的小向导 RNA(sgRNA)进行微注射的胚胎。注射后的胚胎在群体外饲养。第一批幼虫出现后，所有的胚胎和幼虫都被转移到一个带有几个护理工蚁的巢箱中进行进一步发育。该方案适用于诱导突变以分析蚂蚁的特定等级生理和社会行为，但也可能适用于更广泛的膜翅目昆虫和其他昆虫。
