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Significance Colorectal cancer (CRC) is the third leading cause of cancer-related deaths. Genome sequencing studies have provided comprehensive CRC genomic datasets; however, functional validation for most candidate CRC driver genes 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. These studies showedAcvr1b ,Acvr2a , andArid2 could function as tumor suppressors in CRC. We also show that co-occurrent mutations in receptors for activin and transforming growth factor-β synergistically promote tumorigenesis, and shed light on the role of activin receptors. This experimental system can also be applied to organoids derived from other organs, which could further contribute to identification of novel cancer driver genes.

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