CRISPR/Cas9-Induced Genetic Disruption and Characterization of UDP-Galactopyranose Mutase In Trypanosoma Cruzi

The protozoan parasite, Trypanosoma cruzi, is the causative agent of Chagas disease (ChD), or American trypanosomiasis, which affects 6-8 million people in Latin America. It is estimated that 2-3 million people will develop severe lifelong cardiac and/or digestive disorders. ChD has become a life threat not only to endemic regions but most recently also to nonendemic regions, including the United States, owing to extensive worldwide migration in recent years. The lack of a vaccine and the limited efficacy of the two drug treatments available make it urgent to develop novel therapies to treat such a threatening disease. UDP-Galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). In T. cruzi, Galf is found on O-linked oligosaccharides of glycosylphosphatidylinositol (GPI)-anchored mucin glycoproteins and glycoinositolphospholipids (GIPLs), which are highly abundant surface molecules involved in parasite virulence (mucins) and attachment to the insect-vector (GIPLs). Galf is found in several pathogenic organisms but not in humans, thus making the elucidation of its biosynthetic pathway attractive for the development of more effective drugs or vaccines for ChD. The limited genetic tools available to study T. cruzi remains a limiting factor to generate deletion mutants. Here, we propose employing the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system to knockout T. cruzi UGM and identify its essentiality in parasite survival and/or virulence. To this end, we employed the Cas9/sgRNA/pTREX-n attached to a green fluorescent reporter. A donor DNA containing the tag sequence and a marker for antibiotic resistance (blasticidin) were employed to repair the double strand break by homologous recombination. After 7 weeks of transfection, we demonstrated that disruption of UGM is not essential for the survival of T. cruzi. These experiments will further our understanding about T. cruzi UGM, thus, providing potential targets that can be considered in the future development of new drugs and/or vaccines for ChD