Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneity between patients. To facilitate high-throughput genetic testing and functional identification of tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in human colon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigm to establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screening in 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) library in pre-malignant organoids with APC-/-;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complex tumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediators of CRC growth. gRNAs were validated in a secondary screen using unique molecular identifiers (UMIs) to adjust for clonal drift and to distinguish clone size and abundance. Together, these findings highlight a powerful organoid-based platform for pooled CRISPR-Cas9 screening for patient-specific functional genomics.

中文翻译：

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汇集的体外和体内CRISPR-Cas9筛选可鉴定人结肠器官中的肿瘤抑制因子。

大肠癌（CRC）的特点是患者之间的显着遗传和表型异质性。为促进高通量遗传测试和肿瘤驱动程序的功能鉴定，我们开发了一个平台，用于在人类结肠类器官中汇集CRISPR-Cas9筛选。使用转化生长因子β（TGF-β）抗性作为建立体外敏感性和可扩展性的范例，我们确定了在3D类器官中筛选的最佳条件和严格的指导RNA（gRNA）要求。然后，我们在具有APC-/-; KRASG12D突变的恶性前类器官中筛选了全癌肿瘤抑制基因（TSG）文库，将其异种移植以研究复杂肿瘤微环境中的克隆优势。我们确定TGFBR2是最普遍的TSG，其次是CRC生长的已知和以前未知的介体。在二级筛选中使用唯一的分子标识符（UMI）对gRNA进行了验证，以调整克隆的漂移并区分克隆的大小和丰度。总之，这些发现突出了一个强大的基于类器官的平台，可用于针对患者特定功能基因组学的混合CRISPR-Cas9筛选。