Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as “FnCpf1”) protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase–thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

艾美耳球虫球虫属是居住在肠上皮细胞内的细胞内寄生虫，可引起家禽球虫病并导致家禽业的重大经济损失。基因组编辑艾美耳球虫对疫苗和药物的开发具有重要意义。CRISPR/Cas9 已被用于操纵基因组艾美球虫 (E.tenella）。Cas9 的异位表达，即，通过质粒，将引入转基因，这大大限制了其应用，特别是在疫苗开发方面。在这项研究中，我们最初优化了转染协议的条件。我们证明了在优化条件下，FnCas12a（也称为“FnCpf1”）蛋白和 crRNA 靶向的转染乙组氨酸 H4 触发 DNA 双链断裂 体内. 然后我们使用这种策略敲入增强型黄色荧光蛋白的编码盒（EYFP) 和二氢叶酸还原酶-胸苷酸合酶基因 (DHFR) 作为标记内源性的选择标记 肌动蛋白. 设计的E.tenella 寄生虫拥有 EYFP表达在其整个生命周期中。我们的结果表明 FnCas12a 可以触发基因组编辑E.tenella，这增加了基因功能解剖和抗球虫药物和疫苗开发的适用性 艾美耳球虫 物种。