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Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

Abstract

Human intestinal tissue–derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR–Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR–Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2–small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2–3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

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Fig. 1: The steps of lentivirus packaging and transduction.
Fig. 2: The 293FT cells during lentivirus packaging.
Fig. 3: HIE morphologies during transduction and puromycin selection.
Fig. 4: The selection criteria for single-cell HIE clones.
Fig. 5: The comparison between KO and wild-type sequencing results.
Fig. 6: STAT1-KO HIE cells are more susceptible to GII.3 infection.

Data availability

The data are available from the authors upon request. Source data from Fig. 6 were generated using this protocol and were published in ref. 12. Source data are provided with this paper.

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Acknowledgements

We thank B. Lee and the Cell-Based Assay Screening Service (C-BASS) Core in BCM for lentivirus packaging plasmids. This work was supported in part by Public Health Service grants PO1AI057788, U19AI116497, U19AI144297, P30DK56338 and contract HHSN2722017000381 from the National Institutes of Health, by The Cancer Prevention Institute of Texas (CPRIT) RP160283—Baylor College of Medicine Comprehensive Cancer Training Program and RP17005, by NIH P30 shared resource grant CA125123, NIEHS grants P30ES030285 and P42ES0327725, and by the John S. Dunn Research Foundation.

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Authors

Contributions

S.-C.L. and K.H. designed the experiments. S.-C.L. developed the protocol with X.-L.Z. S.-C.L. and X.-L.Z. performed the experiments. S.-C.L., K.H. and M.K.E. wrote the protocol.

Corresponding author

Correspondence to Mary K. Estes.

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Nature Protocols thanks Devanjali Dutta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Lin, S. C. et al. Proc. Natl Acad. Sci. USA 117, 23782–23793 (2020): https://doi.org/10.1073/pnas.2010834117

Haga, K. et al. mBio 11, e00251-20 (2020): https://doi.org/10.1128/mBio.00251-20

Supplementary information

Source data

Source Data Fig. 6

Unprocessed western blots for Fig. 6a and original images for Fig. 6b.

Source Data Fig. 6

Statistical source data for Fig. 6c.

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Lin, SC., Haga, K., Zeng, XL. et al. Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning. Nat Protoc 17, 1004–1027 (2022). https://doi.org/10.1038/s41596-021-00669-0

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