Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland; Department of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
Cell Stem Cell. 2020 Mar 5;26(3):431-440.e8. doi: 10.1016/j.stem.2020.02.007.
Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.
基于全基因组 CRISPR 文库的正向遗传学筛选是解析细胞回路和信号通路的有力工具。然而，将该技术应用于类器官一直受到技术限制的阻碍。在这里，我们通过在单个类器官中捕获 sgRNA 整合，报告了提高汇集文库 CRISPR 筛选准确性和稳健性的方法，这大大减少了全基因组筛选所需的细胞数量。我们将我们的方法应用于野生型和 APC 突变的人肠道类器官，以鉴定参与 TGF-β 介导的生长受限抗性的基因，这是结直肠癌进展过程中的一个关键过程，并验证了包括肿瘤抑制性 SWI/SNF 染色质重塑复合物的多个亚基在内的命中靶点。这些基因中的突变需要 APC 的同时失活以促进 TGF-β 抗性并减弱 TGF-β 靶基因转录。我们的方法可以应用于各种测定和类器官类型，以促进原发性 3D 组织模型中的生物学发现。
