一种用于在结直肠癌细胞来源的类器官中高效进行 CRISPR-Cas9 介导基因敲入的方案。

Department of Cell Biology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.

Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.

STAR Protoc. 2021 Sep 16;2(4):100780. doi: 10.1016/j.xpro.2021.100780. eCollection 2021 Dec 17.

Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of the potential cancer stem cell marker gene, which allows us to investigate their potential for self-replication and pluripotency. We describe the procedure for generating mutant PDOs and their application for stem cell research. For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021).

患者来源的类器官（PDOs）重现了原始结直肠肿瘤组织的细胞异质性。在这里，我们描述了一种生成遗传修饰的 PDO 以研究癌症干细胞的方案。该方案使用 CRISPR-Cas9 系统将 IRES-EGFP-P2A-iCaspase9 盒敲入潜在癌症干细胞标记基因的 3'UTR，使我们能够研究它们的自我复制和多能性潜力。我们描述了生成突变 PDO 及其在干细胞研究中的应用的过程。有关该方案的生成和使用的完整详细信息，请参阅 Okamoto 等人。Okamoto 等人。（2021 年）。