Expanding the tool box for genetic manipulation of Trypanosoma cruzi.

Abstract Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, an illness that affects 6–7 million people and for which there is no effective drug therapy or vaccine. The publication of its complete genome sequence allowed a rapid advance in molecular studies including in silico screening of genes involved with pathogenicity as well as molecular targets for the development of new diagnostic methods, drug therapies and prophylactic vaccines. Alongside with in silico genomic analyses, methods to study gene function in this parasite such as gene deletion, overexpression, mutant complementation and reporter gene expression have been largely explored. More recently, the use of genome-wide strategies is producing a shift towards a global perspective on gene function studies, with the examination of the expression and biological roles of gene networks in different stages of the parasite life cycle and under different contexts of host parasite interactions. Here we describe the molecular tools and protocols currently available to perform genetic manipulation of the T. cruzi genome, with emphasis on recently described strategies of gene editing that will facilitate large-scale functional genomic analyses. These new methodologies are long overdue, since more efficient protocols for genetic manipulation in T. cruzi are urgently needed for a better understanding of the biology of this parasite and molecular processes involved with the complex and often harmful, interaction with its human host.