A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids

Takuya Okamoto; Yasuko Natsume; Hitomi Yamanaka; Mayuko Fukuda; Ryoji Yao

doi:10.1016/j.xpro.2021.100780

A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids

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

Authors

Takuya Okamoto^{1

2}, Yasuko Natsume¹, Hitomi Yamanaka¹, Mayuko Fukuda¹, Ryoji Yao¹

Affiliations

¹ Department of Cell Biology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.
² Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.

Abstract

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).

Keywords: CRISPR; Cancer; Organoids; Stem cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics*
Colorectal Neoplasms* / genetics
Colorectal Neoplasms* / pathology
Gene Editing / methods*
Gene Knock-In Techniques / methods*
Humans
Neoplastic Stem Cells / cytology
Organoids / pathology*
Tumor Cells, Cultured