title: Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.
abstract: Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Sumiti
authors.initials: S
authors.lastname: Vinayak
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Mattie C
authors.initials: MC
authors.lastname: Pawlowic
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Sateriale
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Carrie F
authors.initials: CF
authors.lastname: Brooks
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Caleb J
authors.initials: CJ
authors.lastname: Studstill
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Yael
authors.initials: Y
authors.lastname: Bar-Peled
authors.affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Cipriano
authors.affiliation: 1] Center for Tropical and Emerging Global Diseases, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA [2] Department of Cellular Biology, University of Georgia, Paul D. Coverdell Center, 500 D.W. Brooks Drive, Athens, Georgia 30602, USA.
authors.forename: Boris
authors.initials: B
authors.lastname: Striepen
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003457:Cryptosporidiosis / Q000150:complications / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D003967:Diarrhea / Q000150:complications / Q000469:parasitology*; D004353:Drug Evaluation, Preclinical; D004351:Drug Resistance; D005260:Female; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007422:Intestines / Q000469:parasitology; D051379:Mice; D023421:Models, Animal; D034101:Sporozoites; D013937:Thymidine Kinase / Q000172:deficiency / Q000235:genetics; D014162:Transfection / Q000379:methods; D014295:Trimethoprim / Q000494:pharmacology
chemical_list: D000617:Aminoglycosides; D000962:Antimalarials; D014295:Trimethoprim; D013937:Thymidine Kinase