Journal Article

CRISPR-Cas9–mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes

Takegami, Yujiro, Oshima, Masanobu, Oshima, Hiroko, Park, Jun-Won, Jenkins, Nancy A., Nakayama, Mizuho, Ali, Mohamed A. E., Copeland, Neal G., Yamamoto, Daisuke, Takeda, Haruna, Kataoka, Shiho, An, Tadaichi

2019

Overview

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.

• Publisher: National Academy of Sciences
• Subject: Activin receptors / Adenomatous polyposis coli / Animal models / Animals / Biological Sciences / Cancer / Cell Line, Tumor / Colorectal cancer / Colorectal carcinoma / Colorectal Neoplasms - genetics / Colorectal Neoplasms - metabolism / Colorectal Neoplasms - pathology / CRISPR / CRISPR-Cas Systems / Datasets / Gene Expression Profiling / Gene Knockout Techniques / Gene sequencing / Genes / Genomes / Growth factors / Humans / Intestine / Medical Sciences / Metastases / Mice / Mice, Inbred NOD / Mutagenesis / Mutation / Neoplasm Proteins - biosynthesis / Neoplasm Proteins - genetics / Organoids / Organoids - metabolism / Organoids - pathology / Organs / PNAS Plus / Receptors / Sensitizing / Transforming growth factor / Transforming growth factor-b / Tumor suppressor genes / Tumorigenesis / Tumors / Xenografts / Xenotransplantation