Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

中文翻译：

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化脓性链球菌 Cas9 核糖核蛋白递送，可在锥虫和利什曼原虫中实现高效、快速且无标记的基因编辑


动质体是在动植物界的多种宿主中发现的单细胞真核有鞭毛寄生虫。已知它们会导致人类非洲昏睡病（布氏锥虫）、恰加斯病（克氏锥虫）和各种形式的利什曼病（利什曼原虫属），以及几种具有重要经济影响的动物疾病（非洲锥虫，包括刚果锥虫）。了解这些寄生虫的生物学必然意味着操纵其基因组的能力。在这项研究中，我们证明，转染由重组化脓性链球菌Cas9 ( Sp Cas9) 和体外合成的指导 RNA 组成的核糖核蛋白复合物，可以在无标记条件下对锥虫进行快速有效的遗传修饰。该方法已成功开发用于使布氏锥虫、刚果锥虫和利什曼原虫前鞭毛体生命周期各个阶段的候选基因失活、删除和突变。 Sp Cas9 在这些寄生虫中的功能现在为研究这些寄生虫的研究界提供了一种快速有效的基因组编辑方法，不需要质粒构建和抗生素选择，而只需要克隆和克隆的 PCR 筛选。重要的是，这种方法适用于任何野生型寄生虫。