A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi

Abstract

Few genetic tools were available to work with <i>Trypanosoma cruzi</i> until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the <i>glmS</i> ribozyme from <i>Bacillus subtilis</i>, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in <i>Trypanosoma brucei</i>. In this work we used the CRISPR/Cas9 system for endogenously tagging <i>T. cruzi</i> glycoprotein 72 (<i>TcGP72</i>) and vacuolar proton pyrophosphatase (<i>TcVP1</i>) with the active (<i>glmS</i>) or inactive (<i>M9</i>) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth <i>in vitro</i>. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that <i>T. cruzi</i> produces enough levels of endogenous glucosamine 6-phosphate to stimulate the <i>glmS</i> ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in <i>T. cruzi</i> and to validate potential drug targets in this parasite.