Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
Key Laboratory of Zoonosis of Ministry of Agriculture & National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
Vet Res. 2020 Mar 11;51(1):41. doi: 10.1186/s13567-020-00766-0.
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.
柔嫩艾美耳球虫已成为研究原生动物寄生虫生物学和免疫学的有价值的模式生物，这要归功于反向遗传操作平台的建立。在本报告中，我们描述了 CRISPR（成簇的规律间隔的短回文重复序列）/Cas9（内切酶）系统在柔嫩艾美耳球虫中的有效遗传编辑中的应用，并表明 CRISPR/Cas9 系统通过单个向导 RNA 介导特定位点的双链 DNA 断裂。使用该系统，我们通过将红色荧光蛋白插入 EtMic2 的 C 末端成功标记了内源性微线蛋白 2（EtMic2）。我们的结果将 CRISPR/Cas9 介导的遗传修饰系统的用途扩展到柔嫩艾美耳球虫，为靶向研究顶复门寄生虫中的基因功能开辟了新途径。
