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

Shih-Ching Lin, Kei Haga, Xi-Lei Zeng, Mary K. Estes

Published: 2022-02-22 DOI: 10.1038/s41596-021-00669-0

CRISPR-Cas9

genetic knockout

intestinal organoids

lentivirus transduction

single-cell cloning

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Human intestinal tissue–nobreakderived enteroids (HIEs; also called organoids) are a powerful exvivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological
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ACKNOWLEDGMENTS

Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).
Heo, I. et al. Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat. Microbiol. 3, 814–823 (2018).
Middendorp, S. et al. Adult stem cells in the small intestine are intrinsically programmed with their -specific function. Stem Cells 32, 1083–1091 (2014).
Foulke-Abel, J. et al. Human enteroids as an ex-vivo model of host–pathogen interactions in the gastrointestinal tract. Exp. Biol. Med. 239, 1124–1134 (2014).
Rajan, R. et al. Novel segment- and host-specific patterns of enteroaggregative Escherichia coli adherence to human intestinal enteroids. mBio 9, e02419–17 (2018).
In, J. G. et al. Human mini-guts: new insights into intestinal physiology and host–pathogen interactions. Nat. Rev. Gastroenterol. Hepatol. 13, 633–642 (2016).
Zachos, N. C. et al. Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology. J. Biol. Chem. 291, 3759–3766 (2016).
Clevers, H. COVID-19: organoids go viral. Nat. Rev. Mol. Cell Biol. 21, 355–356 (2020).
Geurts, M. H., van der Vaart, J., Beumer, J. & Clevers, H. The organoid platform: promises and challenges as tools in the fight against COVID-19. Stem Cell Rep. 16, 412–418 (2021).
Hares, M. F. et al. Stem cell-derived enteroid cultures as a tool for dissecting host–parasite interactions in the small intestinal epithelium. Parasite Immunol. 43, e12765 (2021).
Ettayebi, K. et al. Replication of human noroviruses in stem cell-derived human enteroids. Science 353, 1387–1393 (2016).
Lin, S. C. et al. Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids. Proc. Natl Acad. Sci. USA 117, 23782–23793 (2020).
Ettayebi, K. et al. New insights and enhanced human norovirus cultivation in human intestinal enteroids. mSphere 6, e01136–20 (2021).
Murakami, K. et al. Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids. Proc. Natl Acad. Sci. USA 117, 1700–1710 (2020).
Haga, K. et al. Genetic manipulation of human intestinal enteroids demonstrates the necessity of a functional fucosyltransferase 2 gene for secretor-dependent human norovirus infection. mBio 11, e00251–20 (2020).
Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. & Nakata, A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429–5433 (1987).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Paques, F. & Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999).
Urnov, F. D., Rebar, E. J., Holmes, M. C., S., H. & Zhang, P. D. Gregory, Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).
Moore, J. K. & Haber, J. E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell Biol. 16, 2164–2173 (1996).
Durai, S. et al. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 33, 5978–5990 (2005).
Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Chang-Graham, A. L. et al. Rotavirus induces intercellular calcium waves through ADP signaling. Science 370, eabc3621 (2020).
Giuliano, C. J., Lin, A., Girish, V. & Sheltzer, J. M. Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9. Curr. Protoc. Mol. Biol. 128, e100 (2019).
Michels, B. E. et al. Human colon organoids reveal distinct physiologic and oncogenic Wnt responses. J. Exp. Med. 216, 704–720 (2019).
Ousterout, D. G. et al. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat. Commun. 6, 6244 (2015).
Shimokawa, M. et al. Visualization and targeting of LGR5+ human colon cancer stem cells. Nature 545, 187–192 (2017).
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).
Artegiani, B. et al. Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing. Nat. Cell Biol. 22, 321–331 (2020).
Hendriks, D., Clevers, H. & Artegiani, B. CRISPR-Cas tools and their application in genetic engineering of human stem cells and organoids. Cell Stem Cell 27, 705–731 (2020).
Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015).
Matano, M. et al. Modeling colorectal cancer using CRISPR–Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).
Teriyapirom, I., Batista-Rocha, A. S. & Koo, B. K. Genetic engineering in organoids. J. Mol. Med. 99, 555–568 (2021).
A. M. Gaebler et al. Universal and efficient electroporation protocol for genetic engineering of gastrointestinal organoids. J. Vis. Exp. http://doi.org/10.3791/60704 (2020).
H. Miyoshi, T. S. Stappenbeck, In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protoc. 8, 2471–2482 (2013).
Drost, J., Artegiani, B. & Clevers, H. The generation of organoids for studying Wnt signaling. Methods Mol. Biol. 1481, 141–159 (2016).
J. F. Van Lidth de Jeude, J. L. Vermeulen, P. S. Montenegro-Miranda, G. R. Van den Brink, J. Heijmans, A protocol for lentiviral transduction and downstream analysis of intestinal organoids. J. Vis. Exp. http://doi.org/10.3791/52531 (2015).
A. Merenda et al. A protocol for multiple gene knockout in mouse small intestinal organoids using a CRISPR-concatemer. J. Vis. Exp. http://doi.org/10.3791/55916 (2017).
Zhang, J., Chen, L., Zhang, J. & Wang, Y. Drug inducible CRISPR/Cas systems. Comput. Struct. Biotechnol. J. 17, 1171–1177 (2019).
Chen, Y. et al. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 17, 233–244 (2015).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR–Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Hay, E. A. et al. An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome. Neuropeptides 64, 101–107 (2017).
Saxena, K. et al. Human intestinal enteroids: a new model to study human rotavirus infection, host restriction, and pathophysiology. J. Virol. 90, 43–56 (2016).
Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).
Jiang, X., Wang, M., Graham, D. Y. & Estes, M. K. Expression self-assembly and antigenicity of the Norwalk virus capsid protein. J. Virol. 66, 6527–6532 (1992).
VanDussen, K. L., Sonnek, N. M. & Stappenbeck, T. S. L-WRN conditioned medium for gastrointestinal epithelial stem cell culture shows replicable batch-to-batch activity levels across multiple research teams. Stem Cell Res. 37, 101430 (2019).
Sastry, L., Johnson, T., Hobson, M. J., Smucker, B. & Cornetta, K. Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods. Gene Ther. 9, 1155–1162 (2002).
Barczak, W., Suchorska, W., Rubiś, B. & Kulcenty, K. Universal real-time PCR-based assay for lentiviral titration. Mol. Biotechnol. 57, 195–200 (2015).
Joung, J. et al. Genome-scale CRISPR–Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).
Loisy, F. et al. Real-time RT-PCR for norovirus screening in shellfish. J. Virol. Methods 123, 1–7 (2005).

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

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