Department of Homeostatic Regulation, Division of Cellular and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871, Japan.
Institute for Molecular and Cellular Regulation, Gunma University, Gunma, 371-8512, Japan.
Sci Rep. 2022 Jul 8;12(1):11628. doi: 10.1038/s41598-022-15972-3.
The African turquoise killifish Nothobranchius furzeri (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the duration of experiments, and we think that these advances will accelerate aging and developmental studies using N. furzeri.
非洲蓝珍珠鱼（Nothobranchius furzeri）是一种用于研究衰老、与年龄相关的疾病和胚胎滞育的有用模式生物。先前已有报道称，可利用 CRISPR/Cas9 介导的基因敲除和 Tol2 转座子介导的转基因技术在非洲蓝珍珠鱼中实现这一目标。然而，这些方法需要时间来生成敲除和转基因鱼。此外，在非洲蓝珍珠鱼中将大的 DNA 片段作为荧光报告构建体插入靶基因的基因敲入技术尚未建立。在这里，我们展示了三重靶向 CRISPR 介导的单基因敲除可有效地产生全身双等位基因敲除，并能够在 F0 代中研究基因功能。此外，我们通过优化 CRISPR/Cas9 系统开发了一种无需杂交即可创建 knock-in 报告基因的非洲蓝珍珠鱼的方法。这些方法大大缩短了实验时间，我们认为这些进展将加速利用非洲蓝珍珠鱼进行衰老和发育研究。
