Efficient genetic engineering of human intestinal organoids using electroporation
Abstract
Gene modification in untransformed human intestinal cells is an attractive approach for studying gene function in intestinal diseases. However, because of the lack of practical tools, such studies have largely depended upon surrogates, such as gene-engineered mice or immortalized human cell lines. By taking advantage of the recently developed intestinal organoid culture method, we developed a methodology for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors. Here we describe a detailed protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix. We also describe how to stably integrate genes via the piggyBac transposon, as well as precise genome editing using the CRISPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.
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For non-viral methods, electroporation results in higher organoid-editing efficiencies.
“For the non-viral methods, electroporation has been found to result in higher organoid-editing efficiencies (Fujii et al., 2015).”
From Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR (2023) · cites this paper as Fujii et al., 2015
The use of the RNP method allowed an almost 3-fold increase in editing efficiency compared to previously published electroporation-based delivery methods using a plasmid-based approach.
“The use of the RNP method allowed an almost 3-fold increase in the editing efficiency compared to previously published electroporation-based delivery methods using a plasmid-based approach (Fujii et al., 2015).”
From Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR (2023) · cites this paper as Fujii et al., 2015
Editing efficiencies of human organoids using non-viral electroporation methods have been only up to 30%.
“However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%”
From Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR (2023) · cites this paper as Fujii et al., 2015
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“Parts of the protocol have been modified from Fujii et al. (2015) for RNP delivery in cells using electroporation.”
Influential
- Authors
- Fujii, M., Matano, M., Nanki, K., & Sato, T.
- Affiliations
- Keio University (JP)
- Corresponding author
- Sato, Toshiro
- Venue
- Nature Protocols
- Published
- 2015-09-03
- Volume
- 10
- Issue
- 10
- Pages
- 1474-1485
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2159582900
- Cited by
- 364
- References
- 12
- Citations by year
- 2026: 32 · 2025: 42 · 2024: 41 · 2023: 28 · 2022: 57 · 2021: 41 · 2020: 49 · 2019: 19 · 2018: 25 · 2017: 21 · 2016: 8
- FWCI
- 6.94
- Type
- journal article
- Language
- en
- Update notices
- correction (2018-12-05)
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- intestinal organoids, colonic organoids, electroporation, piggyBac transposon, CRISPR/Cas9 genome editing, gene delivery, Matrigel, organoid culture, intestinal diseases
- MeSH terms
- Animals, Cell Line, Flow Cytometry, Genetic Engineering, Humans, Intestines, Organoids, Electroporation, Cell Culture Techniques, Mice
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