Leishmania parasites 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 in Leishmania because 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 Beneke et al. and the glmS ribozyme-based knock-down system in the model parasite Leishmania 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 of L. tarentolae in standard BHI medium resulted in a constitutive down-regulation of an episomal mCherry-glmS reporter by 40 to 60%. For inducible knock-downs, we evaluated the growth of L. tarentolae in 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 episomal mCherry-glmS reporter by more than 70%. However, chromosomal glmS-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 in L. tarentolae as well as the limitations of the glmS system, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.

利什曼原虫寄生虫包括重要的病原体和模型生物，甚至被用于生产重组蛋白。然而，在利什曼原虫中，功能基因组学和必需基因的表征通常受到限制，这是因为用于基因破坏或标记的低通量技术以及缺少用于RNA干扰的成分。在这里，我们测试了Beneke等人的T7 RNA聚合酶依赖性CRISPR-Cas9系统。和基于寄生虫利什曼原虫塔伦托雷的基于glmS核酶的敲低系统。我们成功删除了两个编码鞭毛运动因子Pf16和挽救途径腺嘌呤磷酸核糖基转移酶的参考基因，分别导致了运动性和耐药性寄生虫。相反，我们无法破坏编码线粒体黄素蛋白Erv的基因。在标准BHI培养基中培养塔伦特氏乳杆菌导致游离mCherry-glmS报告基因组成性下调40％至60％。对于诱导敲除，我们评估了替代培养基中塔伦特氏乳杆菌的生长，并确定了补充的MEM，IMDM和McCoy's 5A培养基为候选。在补充的MEM中进行培养可诱导游离葡萄糖的浓度依赖性下调mCherry-glmS记者人数增加了70％以上。但是，编码Pf16，腺嘌呤磷酸核糖基转移酶或Erv的基因的染色体glmS标记未显示敲低表型。我们的数据表明CRISPR-Cas9系统适用于塔伦特氏菌基因的破坏和标签化以及glmS系统的局限性，后者仅限于中等效率的游离敲除，并且未导致染色体的可检测表型击倒。