Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
Nat Genet. 2018 Mar;50(3):460-471. doi: 10.1038/s41588-018-0054-7. Epub 2018 Feb 19.
Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.
初级纤毛组织 Hedgehog 信号转导并塑造胚胎发育，其失调是纤毛病的统一原因。我们通过工程化抗生素选择 Hedgehog 反应细胞并应用全基因组 CRISPR 介导的基因敲除，进行了 Hedgehog 信号转导的功能基因组筛选。该筛选可以稳健地识别具有较少假阳性或假阴性的纤毛信号传导所需的因素。命中基因的特征揭示了几个纤毛结构的新成分，包括含有 δ-微管蛋白和 ε-微管蛋白的蛋白质复合物，该复合物是中心体维持所必需的。该筛选还为分类纤毛病提供了一个无偏工具，并表明许多先天性心脏疾病是由于纤毛信号转导丧失引起的。总之，我们的研究使纤毛功能和纤毛病的系统分析成为可能，并且还定义了一个通过基于 CRISPR 的筛选来解析信号通路的多功能平台。
