Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Efficient genetic engineering of human intestinal organoids using electroporation

An Author Correction to this article was published on 05 December 2018

This article has been updated

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: An overview of gene delivery to human intestinal organoids.
Figure 2: Organoid-forming competency of single-cell dissociated organoids.
Figure 3: Comparison of gene-delivery methods and conditions.
Figure 4: An experimental protocol for electroporation to human intestinal organoids.
Figure 5: Flow cytometry analysis of GFP expression after delivery of a GFP-expressing piggyBac vector with optimized electroporation protocol.
Figure 6: Images of the organoids during selection.

Change history

  • 05 December 2018

    The version of this paper originally published shows incorrect units for two plasmid concentrations. In the "Reagent Setup" section, the instructions for sgRNA-Cas9 plasmid should read "Adjust the concentration of each plasmid to 1 μg μl–1,” rather than "to 1 μg ml–1.” Similarly, all concentrations in the tables in Steps 49A, 49C, and 49D should be in μg μl–1 instead of μg ml–1. Please note that these units have not been corrected in the PDF and HTML versions of the protocol available online.

References

  1. 1

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Koo, B.K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods 9, 81–83 (2012).

    CAS  Article  Google Scholar 

  4. 4

    Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).

    CAS  Article  Google Scholar 

  6. 6

    Wang, F. et al. Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay. Gastroenterology 145, 383–395 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Melkonyan, H., Sorg, C. & Klempt, M. Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). Nucleic Acids Res. 24, 4356–4357 (1996).

    CAS  Article  Google Scholar 

  8. 8

    Ding, S. et al. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122, 473–483 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015).

    CAS  Article  Google Scholar 

  10. 10

    Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  Article  Google Scholar 

  11. 11

    Schwank, G., Andersson-Rolf, A., Koo, B.K., Sasaki, N. & Clevers, H. Generation of BAC transgenic epithelial organoids. PLoS ONE 8, e76871 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), by a Grant-in-Aid for Scientific Research on Innovative Areas 'Stem Cell Aging and Disease' and by Grants-in-Aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology of Japan. L-Wnt3A cells and the R-spondin1–producing cell line were kindly gifted by H. Clevers (Hubrecht Institute) and C. Kuo (Stanford University), respectively.

Author information

Affiliations

Authors

Contributions

M.F., M.M. and K.N. performed the experiments. T.S. conceived and designed the project. M.F. wrote the manuscript.

Corresponding author

Correspondence to Toshiro Sato.

Ethics declarations

Competing interests

T.S. is an inventor on patents involving the organoid culture system (WO/2010/090513 and WO/2012/168930).

Integrated supplementary information

Supplementary Figure 1 Reduced growth efficiency by continuous treatment with CHIR99021.

Images of organoids treated with CHIR99021 for 7 days (left) and without CHIR99021 (with Wnt3A and R-Spondin1)(right) after single cell dissociation. Scale bars, 500 µm.

Supplementary Figure 2 Diagram of the comparisons for optimization of the electroporation method.

The conditions with red characters are superior to the others, and the protocol was optimized by combination of these conditions.

Supplementary Figure 3 Effect of filtration of the dissociated organoids.

Images of dissociated organoids before (left) and after (right) passing through a filter with 20-µm pores. Scale bars, 200 µm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 724 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fujii, M., Matano, M., Nanki, K. et al. Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10, 1474–1485 (2015). https://doi.org/10.1038/nprot.2015.088

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing