CRISPR/Cas9技术在构建荧光标记柔嫩艾美耳球虫β-微管蛋白敲入虫株上的应用OA北大核心CSTPCD

In order to evaluate the application of CRISPR/Cas9 plasmid of Toxoplasma gondii in gene tapping of Eimeria tenella,Etβ-tubulin was used as the target gene.gRNA was designed according to the sequence of Etβ-tubulin gene,and gene-edited plasmids containing gRNA and homologous recombinant plasmids containing target genes and mCherry genes were constructed.The gRNA-containing gene editing plasmid and the homologous recombinant plasmid containing the target gene,the DHFR gene and the mCher-ry gene were co-transfected with E.tenella sporozoites,and the transfected sporozoites were inoculat-ed into the cloaca of chicks,and screened by adding pyrimethamine to the feed,to obtain labeled coc-cidia strains expressing exogenous fluorescent proteins.Single oocysts were selected for propaga-tion,and the labeled strains were identified at gene level and protein level by PCR and fluorescence detection.The results showed that a fluorescent-labeled β-tubulin strain of E.tenella was successfully obtained through single oocyst screening and identification.The results showed that CRISPR/Cas9 tech-nology could be applied to gene knocking in E.tenella,and it also laid a foundation for further study of the function of Et β-tubulin in the parasite.