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

Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genet-ically 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 inves-tigate their potential for self-replication and pluripotency. We describe the pro-cedure 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).

Automated summary

The protocol involves generating genetically modified PDOs using the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of a potential cancer stem cell marker gene. This allows investigation into the potential for self-replication and pluripotency of these cells in stem cell research.