Honeybee Research Institute, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China.
Honeybee Research Institute, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China
G3 (Bethesda). 2019 May 7;9(5):1759-1766. doi: 10.1534/g3.119.400130.
The honeybee () is an important insect pollinator of wild flowers and crops, playing critical roles in the global ecosystem. Additionally, the honeybee serves as an ideal social insect model. Therefore, functional studies on honeybee genes are of great interest. However, until now, effective gene manipulation methods have not been available in honeybees. Here, we reported an improved CRISPR/Cas9 gene-editing method by microinjecting sgRNA and Cas9 protein into the region of zygote formation within 2 hr after queen oviposition, which allows one-step generation of biallelic knockout mutants in honeybee with high efficiency. We first targeted the gene. Two batches of honeybee embryos were collected and injected with sgRNA and Cas9 protein at the ventral cephalic side and the dorsal posterior side of the embryos, respectively. The gene-editing rate at the ventral cephalic side was 93.3%, which was much higher than that (11.8%) of the dorsal-posterior-side injection. To validate the high efficiency of our honeybee gene-editing system, we targeted another gene, , and injected sgRNA and Cas9 protein at the ventral cephalic side in the third batch. A 100% editing rate was obtained. Sanger sequencing of the TA clones showed that 73.3% (for ) and 76.9% (for ) of the edited current-generation embryos were biallelic knockout mutants. These results suggest that the CRISPR/Cas9 method we established permits one-step biallelic knockout of target genes in honeybee embryos, thereby demonstrating an efficient application to functional studies of honeybee genes. It also provides a useful reference to gene editing in other insects with elongated eggs.
蜜蜂是野生花卉和作物的重要传粉昆虫，在全球生态系统中发挥着关键作用。此外，蜜蜂也是一种理想的社会性昆虫模型。因此，对蜜蜂基因的功能研究具有重要意义。然而，到目前为止，蜜蜂中还没有有效的基因操作方法。在这里，我们报道了一种改进的 CRISPR/Cas9 基因编辑方法，通过在蜂王产卵后 2 小时内将 sgRNA 和 Cas9 蛋白注射到合子形成区域，可以一步高效地产生蜜蜂的双等位基因敲除突变体。我们首先针对 基因。收集了两批蜜蜂胚胎，并分别在胚胎的头腹侧和背后侧注射 sgRNA 和 Cas9 蛋白。头腹侧的基因编辑率为 93.3%，明显高于背后侧的编辑率（11.8%）。为了验证我们的蜜蜂基因编辑系统的高效性，我们在第三批胚胎的头腹侧靶向另一个基因 ，并注射 sgRNA 和 Cas9 蛋白。编辑率达到了 100%。TA 克隆的 Sanger 测序显示，73.3%（针对 ）和 76.9%（针对 ）的当前代胚胎是双等位基因敲除突变体。这些结果表明，我们建立的 CRISPR/Cas9 方法可以在蜜蜂胚胎中一步实现靶基因的双等位基因敲除，从而有效地应用于蜜蜂基因的功能研究。它还为其他具有长形卵的昆虫的基因编辑提供了有用的参考。
