Preassembled Single-Stranded RNA-Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium

• Published in: The Journal of infectious diseases Vol. 213; no. 8; pp. 1307 - 1314
• Main Authors: Castellanos-Gonzalez, Alejandro, Perry, Nicolas, Nava, Samantha, White, A. Clinton
• Format: Journal Article
• Language: English
• Published: United States Oxford University Press 15.04.2016
• Subjects: Argonaute Proteins - genetics; Argonaute Proteins - metabolism; Cryptosporidiosis - parasitology; Cryptosporidium; Cryptosporidium - genetics; Cryptosporidium - metabolism; Gene Silencing; Host-Parasite Interactions - genetics; Humans; Major and Brief Reports; PARASITES; RNA, Protozoan - genetics; RNA, Protozoan - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Transfection; Cryptosporidium; siRNA; Cryptosporidium parvum; cryptosporidiosis; silencing; Argonaute
• ISSN: 0022-1899; 1537-6613
• DOI: 10.1093/infdis/jiv588

Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.