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The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, is a new powerful technique for genome editing and has been widely employed to study gene function in a wide variety of organisms.Eimeria tenella, causes coccidiosis in chickens, is a model organism for studying the protozoan parasites and both transient and stable transfection system were development successfully.In the present study, the CRISPR-Cas9 system has been adapted to E.tenella.We introduced the Cas9 nucleaseEYFP into E.tenella and generated guide RNA (gRNA) expression vector by using the E.tenella U6 promoter.E.tenella histone 4 promoter and a 90-bp nuclear localization sequence (NLS) were introduced into the vector to regulate Cas9-EYFP expression.We demonstrated that Cas9 is able to introduce site-specific DNA doublestrand breaks in the E.tenella genome that can be repaired through homologous recombination by red fluorescent protein(RFP) reporter gene knock-in the sporozoites surface antigen 13 (SAG 13) open reading frame (ORF).We selected two specific sites in SAG 13 ORF as the efficiency of reporter gene knock-in the target site is relatively low by CRISPR-Cas9 system.We also introduced TgDHFR gene into the donor plasmid to improve the selection efficiency as TgDHFR was promoted by endogenous SAG 13 promoter which exhibits resistance to pyrimethamine.We cotransfected the E.tenella sporozoites with the Cas9 plasmid (HCEA-U6-sgRNA) and the donor fragment with 700bp homologous repair templates from PCR products using nuclear transfection.We inoculated two-day-old AA broilers with a dosage of 2 × 106 transfected sporozoites via cloacal route.We got a co-expressing EYFP and RFP E.tenella population from fecal samples demonstrated that CRISPR-Cas9 efficient in reporter gene knock-in E.tenella genome.The efficacy of CRISPR-Cas9 and target gene deletion using CRISPR-Cas9 in E.tenella parasite should be further investigated.Our results establish methodologies for introducing desired modifications in the E.tenella genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of Apicomplexan parasites.