Louradour Isabelle, Ghosh Kashinath, Inbar Ehud, Sacks David L, Aluvihare Channa, Harrell Robert A
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
University of Maryland Insect Transformation Facility, The Institute for Bioscience and Biotechnology Research.
J Vis Exp. 2020 Nov 17(165). doi: 10.3791/61924.
Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.
白蛉是利什曼原虫属的天然传播媒介，利什曼原虫是一种原生动物寄生虫，可产生从皮肤损伤到内脏病变等一系列广泛症状。解读传播媒介与寄生虫相互作用的本质对于更好地理解利什曼原虫向宿主的传播至关重要。在控制白蛉传播媒介能力（即携带和传播病原体的能力）的参数中，已表明这些昆虫的内在参数起着关键作用。例如，昆虫免疫反应会影响白蛉对利什曼原虫的传播媒介能力。由于缺乏适用于这些非模式生物的基因表达修饰方法，对这些参数的研究受到了限制。通过小干扰RNA（siRNA）进行基因下调是可行的，但除了技术上具有挑战性外，这种沉默只会导致功能部分丧失，且无法代代相传。CRISPR/Cas9技术介导的靶向诱变最近已应用于巴氏白蛉。该技术可在特定选择的位点产生可遗传的突变，从而能够研究感兴趣的基因。CRISPR/Cas9系统依赖于诱导靶向双链DNA断裂，随后通过非同源末端连接（NHEJ）或同源性驱动修复（HDR）进行修复。NHEJ包括简单地封闭断裂处，通常会导致小的插入/缺失事件。相比之下，HDR利用与目标DNA具有同源性的供体DNA分子作为修复模板。在此，我们介绍一种通过CRISPR/Cas9利用NHEJ进行靶向诱变的白蛉胚胎显微注射方法，这是迄今为止唯一适用于白蛉传播媒介的基因组修饰技术。
