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Updated: Oct 18, 2025
Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids
Published on: February 18, 2020
A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids
Takuya Okamoto1,2, Yasuko Natsume1, Hitomi Yamanaka1
1Department of Cell Biology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, 135-8550, Japan.
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).
Insights
This study presents a method to genetically modify patient-derived organoids (PDOs) using CRISPR-Cas9 technology. These modified PDOs enable the investigation of cancer stem cells
Area of Science:
- Oncology
- Molecular Biology
- Stem Cell Research
Background:
- Patient-derived organoids (PDOs) are valuable models that mimic the cellular diversity of colorectal tumors.
- Investigating cancer stem cells (CSCs) is crucial for understanding tumor recurrence and developing effective therapies.
Purpose of the Study:
- To establish a protocol for generating genetically modified PDOs for CSC research.
- To enable the study of CSC self-replication and pluripotency using a novel genetic modification strategy.
Main Methods:
- Utilized the CRISPR-Cas9 gene-editing system for precise genetic modification.
- Knocked-in an IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of a target CSC marker gene.
- Developed a procedure for generating these mutant PDOs.
Main Results:
- Successfully generated genetically modified PDOs with the integrated cassette.
- The modification allows for the investigation of CSC properties, including self-replication and pluripotency.
Conclusions:
- The described protocol provides a powerful tool for studying cancer stem cells in a relevant preclinical model.
- Genetically modified PDOs offer new avenues for exploring CSC biology and therapeutic targeting in colorectal cancer.
