Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy
Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy
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DOI：
10.1126/scitranslmed.aay0271
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发表时间：
2020-03-25
影响因子：
17.1
通讯作者：
Kunkel, Louis M.
中科院分区：
文献类型：
--
作者：
Lek, Angela;Zhang, Yuanfan;Kunkel, Louis M.
CRISPR-Cas9基因编辑技术和全基因组CRISPR-Cas9文库的出现，使高效、公正的基因筛选成为可能，可以加速遗传疾病治疗发现的进程。在这里，我们展示了全基因组CRISPR-Cas9功能缺失文库的实用性，以确定面部肩周骨肌营养不良症（FSHD）的治疗靶点，FSHD是一种遗传复杂的肌肉营养不良症，目前尚无治疗方法。在FSHD中，遗传和表观遗传变化都会导致DUX4的错误表达，DUX4是FSHD的致病基因，编码高细胞毒性DUX4蛋白。我们进行了全基因组CRISPR-Cas9筛选，以鉴定当DUX4在肌肉细胞中表达时功能丧失导致存活的基因。从我们的筛选中出现的基因阐明了与细胞缺氧反应的致病联系，这被揭示为dux4诱导的细胞死亡的主要驱动因素。低氧信号抑制剂的应用导致DUX4蛋白周转增加，随后减少细胞缺氧反应和细胞死亡。此外，这些化合物被证明成功地减少了患者肌源系中FSHD疾病的生物标志物，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表达途径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见解，并确定了对FSHD具有潜在治疗益处的现有化合物。我们的实验方法为复杂遗传疾病的机制理解和治疗发现提供了一个加速的范例，这可能可以通过成熟的表型选择分析转化为其他疾病。
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.