Gene editing of putative cAMP and Ca²⁺-regulated proteins using an efficient cloning-free CRISPR/Cas9 system inTrypanosoma cruzi

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease, a prominent cause of heart disease in the Americas, for which there is no vaccine or satisfactory treatment available. The life cycle of this protozoan parasite alternates between mammalian and insect hosts, during which, in order to survive, it must adapt to a diversity of environmental conditions it faces. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Cyclic AMP (cAMP) and Calcium (Ca+2) signaling pathways regulate critical cellular processes in this parasite, such as differentiation, osmoregulation, flagellar function, host cell invasion and cell bioenergetics. From the adaptation of CRISPR/Cas9 technology in T. cruzi the reverse genetic approaches for functional analysis of genes have experienced a significant impulse However, the toolbox for genome editing in this parasite still needs to be expanded, for example, to perform multiple gene deletion analyses. Here we used a T7RNAP/Cas9-mediated strategy to tag and delete three genes putatively involved in cAMP and Ca2+ signaling pathways: a putative Ca2+/calmodulin-dependent protein kinase (CAMK), FLAgellar Member 6 (FLAM6) and Cyclic nucleotide-binding domain/C2 domain-containing protein (CC2CP). We were able to endogenously tag these three genes and determine the subcellular localization of the tagged proteins. Furthermore, the strategy used for gene knockout experiments allows us to suggest that TcCC2CP is an essential gene in T. cruzi epimastigotes. Our results obtained in T. cruzi epimastigote forms on these proteins will open new venues for future research.