The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

中文翻译：

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应用全基因组 CRISPR-Cas9 筛选进行面肩肱型肌营养不良症的治疗发现。

CRISPR-Cas9 基因编辑技术和全基因组 CRISPR-Cas9 文库的出现实现了高效、无偏见的基因筛选，可以加速遗传疾病的治疗发现过程。在这里，我们展示了全基因组 CRISPR-Cas9 功能丧失文库的效用，以确定面肩肱型肌营养不良症 (FSHD) 的治疗靶点，FSHD 是一种遗传复杂的肌营养不良症，目前尚无治疗方法。在 FSHD 中，遗传和表观遗传的变化都会导致 DUX4 的错误表达，DUX4 是编码高细胞毒性 DUX4 蛋白的 FSHD 因果基因。我们进行了全基因组 CRISPR-Cas9 筛选，以确定当 DUX4 在肌肉细胞中表达时功能丧失导致存活的基因。从我们的屏幕中出现的基因阐明了与细胞缺氧反应的致病联系，这被证明是 DUX4 诱导的细胞死亡的主要驱动因素。缺氧信号抑制剂的应用导致 DUX4 蛋白周转增加，随后细胞缺氧反应和细胞死亡减少。此外，这些化合物证明成功地减少了患者肌源性细胞系中的 FSHD 疾病生物标志物，并改善了两种 FSHD 斑马鱼模型的结构和功能特性。我们对影响 DUX4 表达的通路的全基因组扰动提供了对 DUX4 诱导发病机制的关键驱动因素的深入了解，并确定了对 FSHD 具有潜在治疗益处的现有化合物。