Wiedeman Justin, Harrison Ruby, Etheridge Ronald Drew
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602.
Department of Cellular Biology, University of Georgia, Athens, GA 30602.
Proc Natl Acad Sci U S A. 2025 Feb 25;122(8):e2416009122. doi: 10.1073/pnas.2416009122. Epub 2025 Feb 18.
While advances in genome editing technologies have simplified gene disruption in many organisms, the study of essential genes requires development of conditional disruption or knockdown systems that are not available in most organisms. Such is the case for , a parasite that causes Chagas disease, a severely neglected tropical disease endemic to Latin America that is often fatal. Our knowledge of the identity of essential genes and their functions in has been severely constrained by historical challenges in very basic genetic manipulation and the absence of RNA interference machinery. Here, we describe the development and use of self-cleaving RNA sequences to conditionally regulate essential gene expression in . Using these tools, we identified essential roles for Polo-like and Aurora kinases in cell division, mirroring their functions in . Importantly, we demonstrate conditional knockdown of essential genes in intracellular amastigotes, the disease-causing stage of the parasite in its human host. This conditional knockdown system enables the efficient and scalable functional characterization of essential genes in and provides a framework for the development of conditional gene knockdown systems for other nonmodel organisms.
虽然基因组编辑技术的进步简化了许多生物体中的基因破坏，但对必需基因的研究需要开发条件性破坏或敲低系统，而大多数生物体中都没有这样的系统。克氏锥虫就是这种情况，它是一种导致恰加斯病的寄生虫，恰加斯病是拉丁美洲特有的一种严重被忽视的热带疾病，通常是致命的。由于在非常基础的基因操作方面存在历史挑战以及缺乏RNA干扰机制，我们对克氏锥虫中必需基因的身份及其功能的了解受到了严重限制。在这里，我们描述了自我切割RNA序列的开发和使用，以条件性调节克氏锥虫中必需基因的表达。使用这些工具，我们确定了Polo样激酶和极光激酶在克氏锥虫细胞分裂中的重要作用，这与它们在其他生物体中的功能相似。重要的是，我们证明了在细胞内无鞭毛体（寄生虫在人类宿主中的致病阶段）中对必需基因的条件性敲低。这种条件性敲低系统能够高效且可扩展地对克氏锥虫中的必需基因进行功能表征，并为开发其他非模式生物的条件性基因敲低系统提供了框架。
