Testing the CRISPR-Cas9 andglmSribozyme systems inLeishmania tarentolae

Abstract

AbstractLeishmaniaparasites include important pathogens and model organisms and are even used for the production of recombinant proteins. However, functional genomics and the characterization of essential genes are often limited inLeishmaniabecause of low-throughput technologies for gene disruption or tagging and the absence of components for RNA interference. Here, we tested the T7 RNA polymerase-dependent CRISPR-Cas9 system by Benekeet al. and theglmSribozyme-based knock-down system in the model parasiteLeishmania tarentolae. We successfully deleted two reference genes encoding the flagellar motility factor Pf16 and the salvage-pathway enzyme adenine phosphoribosyltransferase, resulting in immotile and drug-resistant parasites, respectively. In contrast, we were unable to disrupt the gene encoding the mitochondrial flavoprotein Erv. Cultivation ofL. tarentolaein standard BHI medium resulted in a constitutive down-regulation of an episomalmCherry-glmSreporter by 40 to 60%. For inducible knock-downs, we evaluated the growth ofL. tarentolaein alternative media and identified supplemented MEM, IMDM and McCoy’s 5A medium as candidates. Cultivation in supplemented MEM allowed an inducible, glucosamine concentration-dependent down-regulation of the episomalmCherry-glmSreporter by more than 70%. However, chromosomalglmS-tagging of the genes encoding Pf16, adenine phosphoribosyltransferase or Erv did not reveal a knock-down phenotype. Our data demonstrate the suitability of the CRISPR-Cas9 system for the disruption and tagging of genes inL. tarentolaeas well as the limitations of theglmSsystem, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.