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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.

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella