Department of Biology, University of Maryland, College Park, MD, USA.
Department of Biological Sciences, University of Toronto, Scarborough, ON, Canada.
Sci Rep. 2021 Jul 23;11(1):15138. doi: 10.1038/s41598-021-94577-8.
Direct tests of gene function have historically been performed in a limited number of model organisms. The CRISPR/Cas system is species-agnostic, offering the ability to manipulate genes in a range of models, enabling insights into evolution, development, and physiology. Astatotilapia burtoni, a cichlid fish from the rivers and shoreline around Lake Tanganyika, has been extensively studied in the laboratory to understand evolution and the neural control of behavior. Here we develop protocols for the creation of CRISPR-edited cichlids and create a broadly useful mutant line. By manipulating the Tyrosinase gene, which is necessary for eumelanin pigment production, we describe a fast and reliable approach to quantify and optimize gene editing efficiency. Tyrosinase mutants also remove a major obstruction to imaging, enabling visualization of subdermal structures and fluorophores in situ. These protocols will facilitate broad application of CRISPR/Cas9 to studies of cichlids as well as other non-traditional model aquatic species.
传统上,基因功能的直接检测仅在少数几种模式生物中进行。CRISPR/Cas 系统与物种无关,能够在一系列模型中操纵基因,从而深入了解进化、发育和生理学。伯氏丽鱼(Astatotilapia burtoni)是一种来自坦噶尼喀湖周围河流和海岸的慈鲷鱼,在实验室中被广泛研究,以了解进化和行为的神经控制。在这里,我们开发了用于创建 CRISPR 编辑慈鲷的方案,并创建了一条广泛有用的突变系。通过操纵酪氨酸酶基因,该基因对于真黑色素色素的产生是必需的,我们描述了一种快速可靠的方法来量化和优化基因编辑效率。酪氨酸酶突变体还消除了成像的主要障碍,能够在体内可视化皮下结构和荧光团。这些方案将促进 CRISPR/Cas9 在慈鲷以及其他非传统的水生模式物种研究中的广泛应用。
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