Department of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
Methods Mol Biol. 2022;2488:99-111. doi: 10.1007/978-1-0716-2277-3_8.
The CRISPR/Cas technology has revolutionized forward genetic screening, and thereby facilitated genetic dissection of cellular processes and pathways. TGF-β signaling is a highly conserved cascade involved in development, regeneration, and diseases such as cancer. Even though many core components of the signaling cascade have already been described, several context-dependent pathway modulators remain unknown. To address this knowledge gap, we have recently developed a CRISPR screening approach for identifying TGF-β pathway regulators in three-dimensional organoid culture systems. Here, we provide a detailed protocol describing this approach in human intestinal organoids. With adaptations, this screening method could also be applied to other organoid types, and to other signaling cascades such as EGF or WNT signaling, thereby uncovering important mechanism in regeneration and disease.
CRISPR/Cas 技术彻底改变了正向遗传学筛选，从而促进了细胞过程和途径的遗传解析。TGF-β 信号转导是一种高度保守的级联反应，参与发育、再生和癌症等疾病。尽管信号级联的许多核心成分已经被描述，但几种依赖于上下文的途径调节剂仍然未知。为了解决这一知识空白，我们最近开发了一种 CRISPR 筛选方法，用于在三维类器官培养系统中鉴定 TGF-β 途径调节剂。在这里，我们提供了一个详细的方案，描述了人类肠道类器官中的这种方法。通过适当的调整，这种筛选方法也可以应用于其他类器官类型，以及其他信号级联，如 EGF 或 WNT 信号转导，从而揭示再生和疾病中的重要机制。
