Cheng Peipei, Zhang Zhihao, Yang Fayu, Cai Shuo, Wang Lina, Wang Chunmei, Wang Mi, Liu Yingchun, Fei Chenzhong, Zhang Lifang, Xue Feiqun, Gu Feng
Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
Front Genet. 2021 Sep 22;12:738746. doi: 10.3389/fgene.2021.738746. eCollection 2021.
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 is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of (). Ectopic expression of Cas9, i.e., 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 triggered DNA double-strand breaks . We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein () and dihydrofolate reductase-thymidylate synthase gene () as a selection marker to tag endogenous . The engineered parasite possesses expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in , which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for species.
该物种是寄生于肠道上皮细胞内的细胞内寄生虫，可导致家禽球虫病，并给家禽业造成重大经济损失。对该物种进行基因组编辑对于疫苗和药物的开发至关重要。CRISPR/Cas9已被用于操纵该物种的基因组。Cas9的异位表达，即质粒，会引入转基因，这极大地限制了其应用，尤其是在疫苗开发方面。在本研究中，我们首先优化了转染方案的条件。我们证明，在优化条件下，靶向该物种的FnCas12a（也称为“FnCpf1”）蛋白和crRNA的转染会引发DNA双链断裂。然后，我们使用该策略敲入一个编码增强型黄色荧光蛋白（EYFP）的编码盒和二氢叶酸还原酶-胸苷酸合成酶基因（DHFR-TS）作为选择标记，以标记内源性该物种。经过基因工程改造的该物种寄生虫在其整个生命周期中都有EYFP表达。我们的结果表明，FnCas12a可以在该物种中引发基因组编辑，这增强了剖析基因功能以及开发针对该物种的抗球虫药物和疫苗的适用性。
