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Eimeria species are pathogenic protozoa with a wide range of hosts and the cause of poultry coccidiosis, which results in huge economic losses to the poultry industry. These parasites encode a genome of ∼8000 genes that control a highly coordinated life cycle of asexual replication and sexual differentiation, transmission, and virulence. However, the function and physiological importance of the large majority of these genes remain unknown mostly due to the lack of tools for systematic analysis of gene functions. Here, we report the first application of CRISPR-Cas9 gene editing technology in Eimeria tenella for analysis of gene function at a single gene level as well as for systematic functional analysis of an entire gene family. Using a transgenic line constitutively expressing Cas9, we demonstrated successful and efficient loss of function through non-homologous end joining as well as guided homologous recombination. Application of this approach to the study of the localization of EtGRA9 revealed that the gene encodes a secreted protein whose cellular distribution varied during the life cycle. Systematic disruption of the ApiAp2 transcription factor gene family using this approach revealed that 23 of the 33 factors expressed by this parasite are essential for development and survival in the host. Our data thus establish CRISPR-Cas9 as a powerful technology for gene editing in Eimeria and will set the stage for systematic functional analysis of its genome to understand its biology and pathogenesis, and will make it possible to identify and validate new targets for coccidiosis therapy.

Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite., Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction—a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCE Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Apicomplexan parasites are protozoan organisms that are characterised by complex life cycles and they include medically important species, such as the malaria parasite Plasmodium and the causative agents of toxoplasmosis (Toxoplasma gondii) and cryptosporidiosis (Cryptosporidium spp.). Apicomplexan parasites can infect one or more hosts, in which they differentiate into several morphologically and metabolically distinct life cycle stages. These developmental transitions rely on changes in gene expression. In the last few years, the important roles of different members of the ApiAP2 transcription factor family in regulating life cycle transitions and other aspects of parasite biology have become apparent. Here, we review recent progress in our understanding of the different members of the ApiAP2 transcription factor family in apicomplexan parasites.