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

Few genetic tools were available to work with 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 ribozyme from , which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in . In this work we used the CRISPR/Cas9 system for endogenously tagging glycoprotein 72 () and vacuolar proton pyrophosphatase () with the active () or inactive () 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 . 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 produces enough levels of endogenous glucosamine 6-phosphate to stimulate the ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in and to validate potential drug targets in this parasite.