Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC, United States.
Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, DC, United States.
Elife. 2019 Jan 15;8:e41337. doi: 10.7554/eLife.41337.
CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.
CRISPR/Cas9 基因组编辑在扁形动物门的物种中尚未被报道。我们通过靶向 ω1 来验证这一方法，作为原理的证明。这种分泌型核糖核酸酶对于 Th2 极化和肉芽肿形成至关重要。通过电穿孔或慢病毒颗粒转导将 Cas9 复合物与靶向 ω1 的向导 RNA 暴露于血吸虫卵中。一些卵也用单链供体模板进行转染。从基因编辑寄生虫中扩增子的序列显示 Cas9 催化的突变，包括同源重组修复等位基因，其他分析显示 ω1 转录物和核糖核酸酶耗竭。基因编辑卵未能在巨噬细胞/T 细胞共培养物中极化 Th2 细胞因子反应，而在尾静脉注射到小鼠后，围绕 ω1 突变卵的肺肉芽肿体积大大减少。ω1 的敲除和暴露后这些细胞因子水平的降低展示了编程基因编辑在血吸虫中的功能基因组学的新应用。
