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Review
. 2022 Apr;17(4):1004-1027.
doi: 10.1038/s41596-021-00669-0. Epub 2022 Feb 23.

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

Shih-Ching Lin  1 , Kei Haga  2 , Xi-Lei Zeng  1 , Mary K Estes  3   4
Affiliations
Review

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

Shih-Ching Lin et al. Nat Protoc. 2022 Apr.

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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Figures

Fig. 1 ∣
Fig. 1 ∣. The steps of lentivirus packaging and transduction.
The protocol has seven parts: (1) preparation of lentivirus vector with the CRISPR–Cas9 targeting gene of interest; (2) lentivirus packaging; (3) lentivirus transduction of HIEs; (4) HIE recovery and antibiotic selection; (5) single-cell cloning; (6) sequencing verification of single-cell clones; (7) using KO lines to evaluate host gene function in viral replication or other biological processes.
Fig. 2 ∣
Fig. 2 ∣. The 293FT cells during lentivirus packaging.
a, Cells with 70-80% confluency before transfection. b, Cell morphology at day 1 after transfection. c, Cells start to produce lentiviruses and become less attached at day 2 after transfection. 10× objective lens.
Fig. 3 ∣
Fig. 3 ∣. HIE morphologies during transduction and puromycin selection.
a, Single-cell HIEs for lentiviral transduction at day 0. b, Expanded single-cell clones after 2 d of transduction. c,d, Expanded HIE clones 4 d after transduction, which are ready to be selected with puromycin. e, Transduced HIEs treated with puromycin for 4 d. f, Nontransduced HIEs treated with puromycin for 4 d with dying and dead cells. 20× objective lens.
Fig. 4 ∣
Fig. 4 ∣. The selection criteria for single-cell HIE clones.
Different HIE clones can be observed in each well of 96-well plates under a bright-field microscope. a,b, Surviving single-cell clones growing from individual transduced HIEs. c, A surviving and expanding single-cell clone that is ready for passaging. d-f, Dying or dead single-cell clones that need to be excluded. g,h, Multiple surviving clones in one well that need to be excluded as well since they are mixed populations. 20× objective lens.
Fig. 5 ∣
Fig. 5 ∣. The comparison between KO and wild-type sequencing results.
a, According to the sequencing result of the genomic DNA region around the target site compared with the reference wild-type sequence (Ref), HIE clone 1 contains a 49 bp frameshift mutation in both alleles of the target genes, indicating that the clone is a KO clone. b, HIE clone 2 contains no mutation in both alleles of the target genes, indicating that the clone is a wild-type clone.
Fig. 6 ∣
Fig. 6 ∣. STAT1-KO HIE cells are more susceptible to GII.3 infection.
a, Western blots of cell lysates extracted from WT and STA1-KO detected the endogenous STAT1 protein by the protein-specific antibody (indicated by an asterisk) and confirmed protein loss in the isogenic KO culture. b, Gll.3 HuNoV-positive cells (green) were detected after staining with guinea pig anti-HuNoV Ab with nuclei (blue) stained by DAPI. Infected cells in clusters indicate viral spreading (40× objective lens). c, Numbers of infected cells were calculated in six independent images per condition, and each experiment was performed in triplicate (n = 18 in total). Error bars denote standard deviation of the data. Comparisons were made using the two-tailed Student's t-test. *P < 0.05. Figure reproduced with permission from ref. .
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Related links

    1. lin SC et al. Proc. Natl Acad. Sci. USA 117, 23782–23793 (2020): 10.1073/pnas.2010834117 - DOI - PMC - PubMed
    1. Haga K et al. mBio 11, e00251–20 (2020): 10.1128/mBio.00251-20 - DOI - PMC - PubMed

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