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CRISPR-Cas9汇集筛选鉴定出人结肠类器官中的肿瘤抑制因子
作者:小柯机器人 发布时间:2020/4/30 19:43:02

德国癌症研究中心(DKFZ)Henner F. Farin研究组利用体内外CRISPR-Cas9汇集筛选,鉴定出人结肠类器官中的肿瘤抑制因子。2020年4月28日,《细胞—干细胞》在线发表了这一成果。

为促进高通量遗传测试和肿瘤驱动程序的功能鉴定,研究人员开发了一个平台,可用于在人类结肠类器官中进行CRISPR-Cas9汇集筛选。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,研究人员确定了在3D类器官中筛选的最佳条件和严格的向导RNA(gRNA)要求。
 
然后,研究人员使用APC-/-在恶性前类器官中筛选了全癌肿瘤抑制基因(TSG)库; KRAS G12D突变被异种移植以研究复杂肿瘤微环境中的克隆优势。研究人员确定TGFBR2是最普遍的TSG,其次是已知和未知的结直肠癌(CRC)生长介导因子。
 
在二级筛选中使用唯一的分子标识符(UMI)对gRNA进行了验证,从而可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汇集CRISPR-Cas9筛选。
 
附:英文原文

Title: Pooled In Vitro and In Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids

Author: Birgitta E. Michels, Mohammed H. Mosa, Barbara I. Streibl, Tianzuo Zhan, Constantin Menche, Khalil Abou-El-Ardat, Tahmineh Darvishi, Ewelina Czonka, Sebastian Wagner, Jan Winter, Hind Medyouf, Michael Boutros, Henner F. Farin

Issue&Volume: 2020-04-28

Abstract: Colorectal cancer (CRC) is characterized by prominent genetic and phenotypic heterogeneitybetween patients. To facilitate high-throughput genetic testing and functional identificationof tumor drivers, we developed a platform for pooled CRISPR-Cas9 screening in humancolon organoids. Using transforming growth factor β (TGF-β) resistance as a paradigmto establish sensitivity and scalability in vitro, we identified optimal conditions and strict guide RNA (gRNA) requirements for screeningin 3D organoids. We then screened a pan-cancer tumor suppressor gene (TSG) libraryin pre-malignant organoids with APC/;KRASG12D mutations, which were xenografted to study clonal advantages in context of a complextumor microenvironment. We identified TGFBR2 as the most prevalent TSG, followed by known and previously uncharacterized mediatorsof 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-Cas9screening for patient-specific functional genomics.

DOI: 10.1016/j.stem.2020.04.003

Source: https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(20)30142-9

期刊信息

Cell Stem Cell:《细胞—干细胞》,创刊于2007年。隶属于细胞出版社,最新IF:21.464
官方网址:https://www.cell.com/cell-stem-cell/home
投稿链接:https://www.editorialmanager.com/cell-stem-cell/default.aspx