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 genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cas...

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Detalles Bibliográficos
Autores principales: Okamoto, Takuya, Natsume, Yasuko, Yamanaka, Hitomi, Fukuda, Mayuko, Yao, Ryoji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8455475/
https://www.ncbi.nlm.nih.gov/pubmed/34585151
http://dx.doi.org/10.1016/j.xpro.2021.100780
Descripción
Sumario: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).

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