恰加斯病是一种媒介传播的热带病,影响着全世界数百万人,没有疫苗或令人满意的治疗方法。它是由原生动物寄生虫克氏锥虫引起的,被认为是从北美到南美特有的。这种寄生虫具有独特的代谢和结构特征,使其成为基础研究的有吸引力的生物。克氏虫的遗传操作在历史上一直具有挑战性,与其他致病性原生动物相比。然而,使用原核CRISPR/Cas9系统进行基因组编辑显着提高了产生基因修饰的克氏囊细胞系的能力,成为在这种寄生虫生命周期的不同阶段进行蛋白质功能研究的有力工具,包括感染性色素动物和细胞内阿马斯泰格。使用我们适用于T.Cruzi的CRISPR/Cas9方法,有可能进行淘汰赛,克氏毛虫基因的互补和原位标记。在我们的系统中,我们用含有Cas9序列和单向导RNA的表达载体共转染克氏毛虫。与供体DNA模板一起通过同源重组促进DNA断裂修复。因此,我们使用单个抗性标记修饰该基因的两个等位基因,获得了突变的epimastigotes的同质种群。锥虫中的线粒体Ca2转运对于形成细胞溶质Ca2增加的动力学至关重要,细胞的生物能学,以及生存力和感染力。在本章中,我们描述了在T.cruzi中实现基因组编辑的最有效方法,以突变细胞系的产生为例,以研究参与钙稳态的蛋白质。具体来说,我们描述了我们已经用于研究参与T.Cruzi的钙信号级联的三种蛋白质的方法:肌醇1,4,5-三磷酸受体(TcIP3R),线粒体钙离子蛋白(TcMCU)和钙敏感丙酮酸脱氢酶磷酸酶(TcPDP),使用CRISPR/Cas9技术作为确定其在能量代谢调节中的作用的方法。
Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional
study of proteins in different stages of this parasite\'s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to
study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the
study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.