Eimeria tenella has emerged as valuable model organism for studying the biology and immunology of protozoan parasites with the establishment of the reverse genetic manipulation platform. In this report, we described the application of CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (endonuclease) system for efficient genetic editing in E. tenella, and showed that the CRISPR/Cas9 system mediates site-specific double-strand DNA breaks with a single guide RNA. Using this system, we successfully tagged the endogenous microneme protein 2 (EtMic2) by inserting the red fluorescent protein into the C-terminal of EtMic2. Our results extended the utility of the CRISPR/Cas9-mediated genetic modification system to E. tenella, and opened a new avenue for targeted investigation of gene functions in apicomplexan parasites.

中文翻译：

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利用CRISPR / Cas9系统对原生动物艾美球虫进行遗传修饰

通过建立反向遗传操作平台，艾美球虫已成为研究原生动物寄生虫的生物学和免疫学的有价值的模式生物。在本报告中，我们描述了CRISPR（聚类的规则间隔的短回文重复序列）/ Cas9（核酸内切酶）系统在大肠杆菌中高效基因编辑的应用，并显示CRISPR / Cas9系统介导了位点特异性双链DNA断裂带有单个指导RNA。使用此系统，我们通过将红色荧光蛋白插入EtMic2的C端，成功标记了内源性微nemene蛋白2（EtMic2）。我们的结果将CRISPR / Cas9介导的基因修饰系统的应用扩展到了大肠杆菌，并为有针对性地研究复合体寄生虫的基因功能开辟了一条新途径。