- A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids.
A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids.
STAR protocols (2021-09-30)
Takuya Okamoto, Yasuko Natsume, Hitomi Yamanaka, Mayuko Fukuda, Ryoji Yao
PMID34585151
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).
MATERIALS
Product Number
Brand
Product Description
Roche
DIG Luminescent Detection Kit, sufficient for 50 blots (10 cm x 10 cm each), kit of 1 (5 components), suitable for hybridization
Sigma-Aldrich
Puromycin dihydrochloride, Ready Made Solution, from Streptomyces alboniger, 10 mg/mL in H2O, suitable for cell culture
Sigma-Aldrich
R-Spondin1 expressing 293T Cell Line, The R-Spondin1-Expressing 293T cell line produces RSPO1, a critical protein used to establish 3D organoid cultures from stomach, small intestine, colon, pancreas, and liver sources.
Roche
PCR DIG Probe Synthesis Kit, sufficient for 25 reaction (50 μL final reaction volume)