Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning
Open accessAbstract
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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Delivery of next-generation CRISPR tools by lentivirus or ribonucleoprotein complexes may be a suitable alternative strategy to DNA mutations in adult stem cells.
“Thus, delivery of next-generation CRISPR tools by lentivirus or ribonucleoprotein complexes may be a suitable alternative strategy to DNA mutations in adult stem cells.54,55,56”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [55]
The Introduction’s ‘Comparison with other genome modification methods’ section describes lentiviral delivery of Cas9 and sgRNA into human intestinal enteroids for genome editing. The paper does not establish ribonucleoprotein-complex delivery or the broader claim about next-generation tools in adult stem cells. Page numbers were not available in our copy. (LLM, conf=0.83)
Experimental — accuracy not yet measuredViral methods have associated biosafety and permanent integration issues, whereas non-viral methods are simpler and minimise the time required for production of viral particles.
“However, viral methods have associated biosafety and permanent integration issues, whereas non-viral methods are simpler, minimising the time required for the production of the viral particles, which can take up to 2-3 months (Lin et al., 2022).”
From Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR (2023) · cites this paper as Lin et al., 2022
The Introduction’s “Comparison with other genome modification methods” section says electroporation avoids the labor and time needed to produce lentivirus. It does not identify biosafety or permanent integration as problems, and says lentiviral transduction can be easier in laboratories without the specified screening methods. The Timing section gives lentivirus packaging and concentration steps lasting days, not 2–3 months (page numbers unavailable). (LLM, conf=0.89)
Experimental — accuracy not yet measuredCRISPR-Cas9 genome engineering via lentiviral transduction requires multiple time-consuming steps including cloning a single guide RNA into a plasmid vector, generation of lentivirus particles and transduction of the target cell.
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.2”
From Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9 (2023) · cites this paper as [2]
The Procedure (Parts 1–3) describes cloning an sgRNA into a lentiviral vector, producing lentivirus, and transducing human intestinal enteroids. The Timing and Procedure sections also show that establishing and verifying a knockout enteroid line takes weeks, but they do not establish that lentiviral genome engineering commonly takes 2–3 months across target-cell types; that timing includes selection and single-cell cloning in this specific protocol. (LLM, conf=0.84)
Experimental — accuracy not yet measuredCRISPR-Cas9 genome engineering via lentiviral transduction commonly takes 2-3 months in total.
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.2”
From Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9 (2023) · cites this paper as [2]
The Timing sections describe sgRNA-vector preparation, lentivirus packaging and transduction, followed by weeks of selection, single-cell cloning and clone verification—consistent with a roughly 2–3-month process for knockout human intestinal enteroid lines. The source does not establish that CRISPR–Cas9 engineering by lentiviral transduction commonly takes that long across other cell types or protocols. The indexed passages have no page numbers. (LLM, conf=0.86)
Experimental — accuracy not yet measuredCRISPR-Cas9 genome engineering via lentiviral transduction is an effective method.
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.2”
From Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9 (2023) · cites this paper as [2]
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Cites
Basic local alignment search tool
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990) · Journal of Molecular Biology · doi:10.1006/jmbi.1990.9999 · Cited by 98,669 · see citing works
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
Self-citationThe new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062 · see citing works
Genome-scale CRISPR-Cas9 knockout screening in human cells
Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., Heckl, D., Ebert, B. L., Root, D. E., Doench, J. G., et al. (2014) · Science · doi:10.1126/science.1247005 · Cited by 5,234 · see citing works
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3437 · Cited by 4,637 · see citing works
Sato, T., Stange, D. E., Ferrante, M., Vries, R. G. J., van Es, J. H., van den Brink, S., van Houdt, W. J., Pronk, A., van Gorp, J., Siersema, P. D., et al. (2011) · Gastroenterology · doi:10.1053/j.gastro.2011.07.050 · Cited by 3,753 · see citing works
Organoid Models and Applications in Biomedical Research
Xinaris, C., Brizi, V., & Remuzzi, G. (2015) · The Nephron journals/Nephron journals · doi:10.1159/000433566 · Cited by 3,517 · see citing works
Genetic screens in human cells using the CRISPR-Cas9 system
Wang, T., Wei, J. J., Sabatini, D. M., & Lander, E. S. (2014) · Science · doi:10.1126/science.1246981 · Cited by 2,856 · see citing works
Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987) · Journal of Bacteriology · doi:10.1128/jb.169.12.5429-5433.1987 · Cited by 2,421 · see citing works
Genome editing with engineered zinc finger nucleases
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010) · Nature Reviews Genetics · doi:10.1038/nrg2842 · Cited by 2,285 · see citing works
Pâques, F., & Haber, J. E. (1999) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.63.2.349-404.1999 · Cited by 2,120 · see citing works
Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752 · see citing works
Schwank, G., Koo, B.-K., Sasselli, V., Dekkers, J. F., Heo, I., Demircan, T., Sasaki, N., Boymans, S., Cuppen, E., van der Ent, C. K., et al. (2013) · Cell Stem Cell · doi:10.1016/j.stem.2013.11.002 · Cited by 1,424 · see citing works
Replication of human noroviruses in stem cell–derived human enteroids
Ettayebi, K., Crawford, S. E., Murakami, K., Broughman, J. R., Karandikar, U., Tenge, V. R., Neill, F. H., Blutt, S. E., Zeng, X.-L., Qu, L., et al. (2016) · Science · doi:10.1126/science.aaf5211 · Cited by 1,305 · see citing works
Self-citationGenome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
Joung, J., Konermann, S., Gootenberg, J. S., Abudayyeh, O. O., Platt, R. J., Brigham, M. D., Sanjana, N. E., & Zhang, F. (2017) · Nature Protocols · doi:10.1038/nprot.2017.016 · Cited by 1,243 · see citing works
Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids
Matano, M., Date, S., Shimokawa, M., Takano, A., Fujii, M., Ohta, Y., Watanabe, T., Kanai, T., & Sato, T. (2015) · Nature Medicine · doi:10.1038/nm.3802 · Cited by 1,135 · see citing works
Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein
Jiang, X., Wang, M., Graham, D. Y., & Estes, M. K. (1992) · Journal of Virology · doi:10.1128/jvi.66.11.6527-6532.1992 · Cited by 856 · see citing works
Self-citationMoore, J. K., & Haber, J. E. (1996) · Molecular and Cellular Biology · doi:10.1128/mcb.16.5.2164 · Cited by 728 · see citing works
Visualization and targeting of LGR5+ human colon cancer stem cells
Shimokawa, M., Ohta, Y., Nishikori, S., Matano, M., Takano, A., Fujii, M., Date, S., Sugimoto, S., Kanai, T., & Sato, T. (2017) · Nature · doi:10.1038/nature22081 · Cited by 728 · see citing works
In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture
Miyoshi, H., & Stappenbeck, T. S. (2013) · Nature Protocols · doi:10.1038/nprot.2013.153 · Cited by 724 · see citing works
Durai, S. (2005) · Nucleic Acids Research · doi:10.1093/nar/gki912 · Cited by 522 · see citing works
Ousterout, D. G., Kabadi, A. M., Thakore, P. I., Majoros, W. H., Reddy, T. E., & Gersbach, C. A. (2015) · Nature Communications · doi:10.1038/ncomms7244 · Cited by 470 · see citing works
Real-time RT-PCR for norovirus screening in shellfish
Loisy, F., Atmar, R. L., Guillon, P., Cann, P. L., Pommepuy, M., & Guyader, F. S. L. (2004) · Journal of Virological Methods · doi:10.1016/j.jviromet.2004.08.023 · Cited by 423 · see citing works
Modelling Cryptosporidium infection in human small intestinal and lung organoids
Heo, I., Dutta, D., Schaefer, D. A., Iakobachvili, N., Artegiani, B., Sachs, N., Boonekamp, K. E., Bowden, G. D., Hendrickx, A. P. A., Willems, R. J. L., et al. (2018) · Nature Microbiology · doi:10.1038/s41564-018-0177-8 · Cited by 386 · see citing works
Saxena, K., Blutt, S. E., Ettayebi, K., Zeng, X.-L., Broughman, J. R., Crawford, S. E., Karandikar, U. C., Sastri, N. P., Conner, M. E., Opekun, A. R., et al. (2016) · Journal of Virology · doi:10.1128/jvi.01930-15 · Cited by 375 · see citing works
Self-citationEfficient genetic engineering of human intestinal organoids using electroporation
Fujii, M., Matano, M., Nanki, K., & Sato, T. (2015) · Nature Protocols · doi:10.1038/nprot.2015.088 · Cited by 364 · see citing works
Middendorp, S., Schneeberger, K., Wiegerinck, C. L., Mokrý, M., Akkerman, R. D. L., Wijngaarden, S., Clevers, H. C., & Nieuwenhuis, E. E. S. (2014) · Stem Cells · doi:10.1002/stem.1655 · Cited by 302 · see citing works
Artegiani, B., Hendriks, D. F. G., Beumer, J., Kok, R. N. U., Zheng, X., Joore, I. P., Lopes, S. M. C. D. S., van Zon, J. S., Tans, S. J., & Clevers, H. C. (2020) · Nature Cell Biology · doi:10.1038/s41556-020-0472-5 · Cited by 302 · see citing works
Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods
Sastry, L., Johnson, T., Hobson, M. J., Smucker, B., & Cornetta, K. G. (2002) · Gene Therapy · doi:10.1038/sj.gt.3301731 · Cited by 296 · see citing works
Zachos, N., Kovbasnjuk, O., Foulke-Abel, J. D., In, J. G., Blutt, S., de Jonge, H. D., Estes, M., & Donowitz, M. (2015) · Journal of Biological Chemistry · doi:10.1074/jbc.r114.635995 · Cited by 261 · see citing works
Self-citationCRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids
Hendriks, D. F. G., Clevers, H. C., & Artegiani, B. (2020) · Cell stem cell · doi:10.1016/j.stem.2020.10.014 · Cited by 196 · see citing works
Foulke-Abel, J., In, J., Kovbasnjuk, O., Zachos, N. C., Ettayebi, K., Blutt, S. E., Hyser, J. M., Zeng, X.-L., Crawford, S. E., Broughman, J. R., et al. (2014) · Experimental Biology and Medicine · doi:10.1177/1535370214529398 · Cited by 179 · see citing works
Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9
Chen, Y., Cao, J., Xiong, M., Petersen, A. J., Dong, Y. W., Tao, Y., Huang, C. T.-L., Du, Z., & Zhang, S. (2015) · Cell stem cell · doi:10.1016/j.stem.2015.06.001 · Cited by 160 · see citing works
Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9
Giuliano, C. J., Lin, A., Girish, V., & Sheltzer, J. M. (2019) · Current Protocols in Molecular Biology · doi:10.1002/cpmb.100 · Cited by 130 · see citing works
New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids
Ettayebi, K., Tenge, V. R., Cortes-Penfield, N. W., Crawford, S. E., Neill, F. H., Zeng, X., Yu, X., Ayyar, B. V., Burrin, D. G., Ramani, S., et al. (2021) · mSphere · doi:10.1128/msphere.01136-20 · Cited by 126 · see citing works
Self-citationHuman mini-guts: New insights into intestinal physiology and host–pathogen interactions
In, J. G., Foulke-Abel, J., Estes, M. K., Zachos, N. C., Kovbasnjuk, O., & Donowitz, M. (2016) · Nature Reviews Gastroenterology & Hepatology · doi:10.1038/nrgastro.2016.142 · Cited by 125 · see citing works
Self-citationMurakami, K., Tenge, V. R., Karandikar, U. C., Lin, S., Ramani, S., Ettayebi, K., Crawford, S. E., Zeng, X., Neill, F. H., Ayyar, B. V., et al. (2020) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1910138117 · Cited by 118 · see citing works
Self-citationVanDussen, K. L., Sonnek, N. M., & Stappenbeck, T. S. (2019) · Stem Cell Research · doi:10.1016/j.scr.2019.101430 · Cited by 117 · see citing works
Haga, K., Ettayebi, K., Tenge, V. R., Karandikar, U. C., Lewis, M. A., Lin, S., Neill, F. H., Ayyar, B. V., Zeng, X., Larson, G., et al. (2020) · mBio · doi:10.1128/mbio.00251-20 · Cited by 110 · see citing works
Self-citationHuman colon organoids reveal distinct physiologic and oncogenic Wnt responses
Michels, B. E., Mosa, M. H., Grebbin, B. M., Yepes, D., Darvishi, T., Hausmann, J., Urlaub, H., Zeuzem, S., Kvasnicka, H. M., Oellerich, T., et al. (2019) · The Journal of Experimental Medicine · doi:10.1084/jem.20180823 · Cited by 87 · see citing works
Lin, S., Qu, L., Ettayebi, K., Crawford, S. E., Blutt, S. E., Robertson, M. J., Zeng, X., Tenge, V. R., Ayyar, B. V., Karandikar, U. C., et al. (2020) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2010834117 · Cited by 87 · see citing works
Self-citationRotavirus induces intercellular calcium waves through ADP signaling
Chang‐Graham, A. L., Perry, J. L., Engevik, M. A., Engevik, K. A., Scribano, F. J., Gebert, J. T., Danhof, H. A., Nelson, J. C., Kellen, J., Strtak, A. C., et al. (2020) · Science · doi:10.1126/science.abc3621 · Cited by 82 · see citing works
Self-citationA Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids
de Jeude, J. F. V. L., Vermeulen, J. L. M., Montenegro-Miranda, P. S., BRINK, G. R. V. D., & Heijmans, J. (2015) · Journal of Visualized Experiments · doi:10.3791/52531 · Cited by 81 · see citing works
Rajan, A., Vela, L., Zeng, X., Yu, X., Shroyer, N. F., Blutt, S. E., Poole, N. M., Carlin, L. G., Nataro, J. P., Estes, M. K., et al. (2018) · mBio · doi:10.1128/mbio.02419-17 · Cited by 80 · see citing works
Self-citationClevers, H. C. (2020) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-020-0258-4 · Cited by 75 · see citing works
Universal Real-Time PCR-Based Assay for Lentiviral Titration
Barczak, W., Suchorska, W. M., Rubiś, B., & Kulcenty, K. I. (2014) · Molecular Biotechnology · doi:10.1007/s12033-014-9815-4 · Cited by 73 · see citing works
Drug Inducible CRISPR/Cas Systems
Zhang, J., Chen, L., Zhang, J., & Wang, Y. (2019) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2019.07.015 · Cited by 71 · see citing works
Genetic engineering in organoids
Teriyapirom, I., Batista-Rocha, A. S., & Koo, B.-K. (2021) · Journal of Molecular Medicine · doi:10.1007/s00109-020-02029-z · Cited by 68 · see citing works
Hay, E. A., Khalaf, A. R., Marini, P., Brown, A., Heath, K., Sheppard, D., & MacKenzie, A. (2016) · Neuropeptides · doi:10.1016/j.npep.2016.11.003 · Cited by 30 · see citing works
The Organoid Platform: Promises and Challenges as Tools in the Fight against COVID-19
Geurts, M. H., van der Vaart, J., Beumer, J., & Clevers, H. C. (2021) · Stem Cell Reports · doi:10.1016/j.stemcr.2020.11.009 · Cited by 26 · see citing works
The Generation of Organoids for Studying Wnt Signaling
Drost, J., Artegiani, B., & Clevers, H. C. (2016) · Methods in molecular biology · doi:10.1007/978-1-4939-6393-5_15 · Cited by 24 · see citing works
Gaebler, A.-M., Hennig, A., Buczolich, K., Weitz, J., Welsch, T., Stange, D. E., & Pape, K. (2020) · Journal of Visualized Experiments · doi:10.3791/60704 · Cited by 18 · see citing works
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
Merenda, A., Andersson-Rolf, A., Mustata, R. C., Li, T., Kim, H., & Koo, B. (2017) · Journal of Visualized Experiments · doi:10.3791/55916 · Cited by 18 · see citing works
Hares, M. F., Tiffney, E., Johnston, L. J., Luu, L., Stewart, C. J., Flynn, R. J., & Coombes, J. L. (2020) · Parasite Immunology · doi:10.1111/pim.12765 · Cited by 16 · see citing works
Flagged first, then by citations
Research works printed but not linked: 2
- [12]Lin SC Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids. Proc. Natl Acad. Sci. USA 117, 23782– 23793 (2020). 32907944 PMC7519316
- [15]Haga K 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). 32184242 PMC7078471
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Xiang, L., Liang, J., Wang, Z., Lin, F. H., Zhuang, Y., Saiding, Q., Wang, F., Deng, L., & Cui, W. (2023) · Science Advances · doi:10.1126/sciadv.adc9375 · Cited by 72 · see citing works
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Zeng, G., Yu, Y., Wang, M., Liu, J., He, G., Yu, S., Yan, H., Yang, L., Li, H., & Peng, X. (2024) · Journal of Translational Medicine · doi:10.1186/s12967-024-05824-1 · Cited by 43 · see citing works
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Ma, L., Yang, S., Peng, Q., Zhang, J., & Zhang, J. (2023) · Gene · doi:10.1016/j.gene.2023.147480 · Cited by 44 · see citing works
New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment
Yan, B., & Liang, Y. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms232415758 · Cited by 28 · see citing works
Surface atom knockout for the active site exposure of alloy catalyst
Ma, Y., Yang, Q., Qi, J., Zhang, Y., Gao, Y., Zeng, Y., Jiang, N., Sun, Y., Qu, K., Fang, W., et al. (2024) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2319525121 · Cited by 26 · see citing works
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Lin, S., Zhao, F. R., Jáňová, H., Gervais, A., Rucknagel, S., Murray, K. O., Casanova, J., & Diamond, M. (2023) · Nature Communications · doi:10.1038/s41467-023-41600-3 · Cited by 24 · see citing works
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Skoufou-Papoutsaki, N., Adler, S., D'Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F., Nayak, K., Russell, A., et al. (2023) · Disease Models & Mechanisms · doi:10.1242/dmm.050279 · Cited by 22 · see citing works
“However, viral methods have associated biosafety and permanent integration issues, whereas non-viral methods are simpler, minimising the time required for the production of the viral particles, which can take up to 2-3 months (Lin et al., 2022).”
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Wu, W., Jiang, Y.-N., Xing, D.-H., Zhai, Y.-X., Sun, H., He, X., Luo, K.-P., Xu, P., Pan, F., Dong, G.-L., et al. (2024) · Cell Death Discovery · doi:10.1038/s41420-024-01940-5 · Cited by 21 · see citing works
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Celotti, M., Derks, L. L. M., van Es, J., van Boxtel, R., Clevers, H., & Geurts, M. H. (2024) · STAR Protocols · doi:10.1016/j.xpro.2024.103189 · Cited by 17 · see citing works
Hou, G., Beatty, W. L., Ren, L., Ooi, Y. S., Son, J., Zhu, Y., Sheng, Q., Huang, W., Li, D., Liu, C., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-59090-w · Cited by 16 · see citing works
Hao, Y., Chen, P., Guo, S., Li, M., Jin, X., Zhang, M., Deng, W., Li, P., Lei, W., Liang, A., et al. (2023) · Frontiers of Medicine · doi:10.1007/s11684-023-1010-1 · Cited by 17 · see citing works
Yang, H., Niu, S., Guo, M.-H., & Xue, Y. (2024) · Archives of Toxicology · doi:10.1007/s00204-024-03777-4 · Cited by 16 · see citing works
Organoids: A new frontier in precision medicine and engineering
Gao, Q., Wu, Y., Mao, Y., Chen, X., Geng, Z., & Su, J. (2025) · Precision medicine and engineering. · doi:10.1016/j.preme.2025.100049 · Cited by 17 · see citing works
MYADM binds human parechovirus 1 and is essential for viral entry
Qiao, W., Richards, C. M., Kim, Y., Zengel, J. R., Ding, S., Greenberg, H. B., & Carette, J. E. (2024) · Nature Communications · doi:10.1038/s41467-024-47825-0 · Cited by 14 · see citing works
Gastrointestinal organoids in the study of viral infections
Gebert, J. T., Scribano, F. J., Engevik, K. A., Perry, J. L., & Hyser, J. M. (2022) · American Journal of Physiology-Gastrointestinal and Liver Physiology · doi:10.1152/ajpgi.00152.2022 · Cited by 13 · see citing works
CRISPR-based functional genomics tools in vertebrate models
Varshney, G. K., & Burgess, S. M. (2025) · Experimental & Molecular Medicine · doi:10.1038/s12276-025-01514-0 · Cited by 14 · see citing works
Waerlop, G., Janssens, Y., Jacobs, B., Jarczowski, F., Diessner, A., Leroux-Roels, G. G., Klimyuk, V. I., Leroux‐Roels, I., & Thieme, F. (2023) · Frontiers in Immunology · doi:10.3389/fimmu.2023.1188431 · Cited by 10 · see citing works
Kaur, G., Crawford, S. E., Cregeen, S. J., Surathu, A., Ayyar, B. V., Apostol, C. V., Nguyen‐Phuc, H., Ettayebi, K., Boussattach, A., Zeng, X., et al. (2026) · Science Advances · doi:10.1126/sciadv.aeb0455 · Cited by 10 · see citing works
Self-citationChan, D. K. H., Collins, S. D., & Buczacki, S. J. A. (2023) · STAR Protocols · doi:10.1016/j.xpro.2022.101978 · Cited by 8 · see citing works
“CRISPR-Cas9 genome engineering via lentiviral transduction is an effective method but requires multiple time-consuming steps including cloning a single guide RNA (sgRNA) into a plasmid vector, generation of lentivirus particles and transduction of the target cell, commonly taking 2–3 months in total.(2)”
MethodIntestinal epithelial TLR5 signaling promotes barrier-supportive macrophages
Tsai, M.-T., Callaghan, R., Ng, C., Peres-Tintin, L., Matthews, D. B., Stanford, N., Ganesh, K., Estes, M. K., & Diehl, G. E. (2026) · Science Immunology · doi:10.1126/sciimmunol.adr4057 · Cited by 7 · see citing works
Self-citationCRISPR-Cas9-Based Technology for Studying Enteric Virus Infection
Hirano, J., Murakami, K., & Hayashi, T. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2022.888878 · Cited by 7 · see citing works
Zhang, N., Liu, F., Zhao, Y., Sun, X., Wen, B., Lu, J., Yan, C., & Li, D. (2024) · The Journal of Pathology · doi:10.1002/path.6255 · Cited by 6 · see citing works
Organoids to Remodel SARS-CoV-2 Research: Updates, Limitations and Perspectives
An, Y., He, Y., Ge, N., Guo, J., Yang, F., & Sun, S. (2023) · Aging and Disease · doi:10.14336/ad.2023.0209 · Cited by 6 · see citing works
Wang, Y., Liu, X., Xuan, W., Huang, W., Zhu, Y., Mao, C., & Liu, Y. (2026) · Molecular Therapy · doi:10.1016/j.ymthe.2026.03.003 · Cited by 5 · see citing works
Pinski, A. N., Gan, T., Lin, S., Droit, L., Diamond, M., Barouch, D. H., & Wang, D. (2024) · Journal of Virology · doi:10.1128/jvi.00043-24 · Cited by 5 · see citing works
Self-citationKahaki, S. A. M., Ebrahimzadeh, N., Fahimi, H., & Moshiri, A. (2024) · PLoS ONE · doi:10.1371/journal.pone.0310368 · Cited by 6 · see citing works
Li, L., Zhang, Z., Wang, X., Zhao, H., Liu, L., Xiao, Y., Hua, S., & Chen, Y. (2025) · Advanced Science · doi:10.1002/advs.202415559 · Cited by 5 · see citing works
Gebert, J. T., Scribano, F. J., Engevik, K. A., & Hyser, J. M. (2024) · Journal of Visualized Experiments · doi:10.3791/66132 · Cited by 5 · see citing works
Modeling gut inflammation using intestinal organoids: Advances, challenges, and future perspectives
Guzauskiene, J., Valentelyte, D., Butaite, G., Kulokiene, U., Laukaitiene, V., Inčiūraitė, R., & Skiecevičienė, J. (2025) · Best Practice & Research Clinical Gastroenterology · doi:10.1016/j.bpg.2025.102048 · Cited by 4 · see citing works
Zhao, X., Gao, J., Zhu, X., Chen, Y., Ge, H., Xiao, Y., Han, Q., Zhao, X., Zhao, X., & Zhao, R. C. (2025) · Stem Cells and Development · doi:10.1089/scd.2025.0021 · Cited by 4 · see citing works
Peng, W., Zeng, C., Xu, J., Zhao, H., Zhu, Q., Xu, H., Chen, H., Huang, H., Zhou, Y., & Zhao, C. (2023) · Journal of Gastroenterology and Hepatology · doi:10.1111/jgh.16311 · Cited by 4 · see citing works
Chen, Y., Ke, Y., Jiang, Z., & Yang, G. (2025) · Biomaterials Science · doi:10.1039/d5bm00194c · Cited by 5 · see citing works
Jobin, C., Harvey, M., Lacouture, A., Weidmann, C., Neveu, B., Pouliot, F., & Audet‐Walsh, É. (2024) · STAR Protocols · doi:10.1016/j.xpro.2024.103200 · Cited by 3 · see citing works
Loss of mucin 2 and MHC II molecules causes rare resistance to murine RV infection
Bomidi, C., Sawyer, F. M., Shroyer, N. F., Conner, M. E., Estes, M. K., & Blutt, S. E. (2024) · Journal of Virology · doi:10.1128/jvi.01507-24 · Cited by 3 · see citing works
Self-citationMagne, L., Bugarin, F., & Ferrand, A. (2025) · Biology of the Cell · doi:10.1111/boc.70003 · Cited by 2 · see citing works
microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2
Du, L., Billaud, J.-N., Telwatte, S., Kadiyala, G. N., Dabral, P., Bouzidi, M. S., Guatelli, J., Yukl, S. A., & Pillai, S. K. (2026) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2026.102844 · Cited by 2 · see citing works
Virus-Like Particles as a CRISPR-Cas9 Delivery Tool
Shindadkar, S., & Severson, K. M. (2024) · Journal of Undergraduate Life Sciences · doi:10.33137/juls.v16i1.42386 · Cited by 2 · see citing works
Liu, Y., Zhu, J., Yu, L., Zhao, Y., Cao, X., Wei, S., Zeng, J., Chen, H., Lu, Z., Chen, B., et al. (2024) · Journal of Colloid and Interface Science · doi:10.1016/j.jcis.2024.12.031 · Cited by 2 · see citing works
Kaur, G., Crawford, S. E., Ramani, S., Ayyar, B. V., Boussattach, A., Zeng, X., Atmar, R. L., Nguyen‐Phuc, H., Coarfa, C., & Estes, M. K. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.05.22.655571 · Cited by 2 · see citing works
Self-citationWilson, A. P., Moshal, K. S., Franca, A., Ramani, S., Gallucci, R. M., Chaaban, H., & Burge, K. Y. (2024) · STAR Protocols · doi:10.1016/j.xpro.2024.103082 · Cited by 1 · see citing works
Han, Y., Wu, K., Xin, P., Fu, Y., Li, W., Ma, J., Jiang, H., & Zhao, X. (2024) · Physiological Reports · doi:10.14814/phy2.70160 · Cited by 1 · see citing works
SGLP: A novel method for mining single-cell specific gene clusters
Xu, Q., Zhang, Y., Li, W., & Zhu, J. (2025) · Biomedical Signal Processing and Control · doi:10.1016/j.bspc.2025.107944 · Cited by 1 · see citing works
Huang, J.-S., Wang, J., Yuan, Y., Zhang, T., Li, B., Zhao, F.-Y., Hao, L., Yu, Z.-W., & Wang, H. (2025) · Frontiers in Immunology · doi:10.3389/fimmu.2025.1648139 · Cited by 1 · see citing works
Organoids: From Bench to Bedside Applications
Li, K.-L., Cao, R., Li, M.-C., Tian, Z.-C., Fan, H.-H., Hong, B., & Liu, X.-J. (2026) · MedComm · doi:10.1002/mco2.70768 · Cited by 1 · see citing works
Zhu, G., Zhao, Y., Chen, M., Lu, S., Xu, L., Chen, G., Zeng, L., & Chen, J. (2026) · Materials Today Bio · doi:10.1016/j.mtbio.2026.103651 · Cited by 1 · see citing works
Exploitation of In Vivo-Emulated In Vitro System in Advanced Food Science
Li, Y., Li, P., Yu, X. H., Zheng, X., & Gu, Q. (2023) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.2c07289 · Cited by 1 · see citing works
Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPRPreprint
Skoufou-Papoutsaki, N., Adler, S., D’Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F. M., Nayak, K., Russell, A., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.03.31.535108 · Cited by 1 · see citing works
Knockout of FGL1 in Tumor Cell Lines Leads to Decreased Binding Between MHC II and LAG 3
Kang, R. (2022) · International Symposium on Artificial Intelligence for Medicine Sciences (ISAIMS) · doi:10.1145/3570773.3570790
Gedik, M. E., Saatci, Ö., Oberholtzer, N. P., Üner, M., Akbulut, Ö., Çetįn, M., Aras, M., İbiş, K., Çalışkan, B., Banoğlu, E., et al. (2024) · doi:10.1158/0008-5472.25735665
Gedik, M. E., Saatci, Ö., Oberholtzer, N. P., Üner, M., Akbulut, Ö., Çetįn, M., Aras, M., İbiş, K., Çalışkan, B., Banoğlu, E., et al. (2024) · doi:10.1158/0008-5472.25735665.v1
Park, S., Cho, S.-G., Chung, H. W., Jin, H. S., Kim, T., & Lee, J. M. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.11.07.687139
Yang, L., Hatterschide, J., Worota, R. E., Cooley, K. A., & Coyne, C. B. (2026) · Nature Protocols · doi:10.1038/s41596-026-01402-5
Nguyen, T. V., Nguyen-Tran, H.-H., Nguyen, T.-N., & Hsu, T. (2026) · International Journal of Molecular Sciences · doi:10.3390/ijms27156994
The CRISPR/Cas technology landscape: From molecular mechanisms to multidisciplinary applications
Liu, Z., Yang, Y., Li, Z., Li, P., Jia, J., Qiao, X., Su, Y., Qin, Y., Wang, H., Dai, Y., et al. (2026) · Genes & Diseases · doi:10.1016/j.gendis.2026.102484
Wang, Y., Li, X., Liu, C., He, B., Liu, Y., Liu, Q., Liu, C., Chen, D., Wang, S., & Zhang, L. (2026) · Journal of Genetic Engineering and Biotechnology · doi:10.1016/j.jgeb.2026.100830
- Authors
- Lin, S.-C., Haga, K., Zeng, X.-L., & Estes, M. K.
- Affiliations
- Baylor College of Medicine (US)
- Kitasato University (JP)
- Corresponding author
- Estes, Mary K.
- Venue
- Nature Protocols
- Published
- 2022-02-23
- Volume
- 17
- Issue
- 4
- Pages
- 1004-1027
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC9059808
- OpenAlex
- W4213458344
- Cited by
- 63
- References
- 55
- Citations by year
- 2026: 10 · 2025: 13 · 2024: 19 · 2023: 15 · 2022: 6
- FWCI
- 1.38
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Commons-TMC (Texas Medical Center) (submittedVersion)
- Funders
- U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (PO1AI057788, U19AI116497, U19AI144297, HHSN2722017000381)
- U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (P30DK56338)
- Cancer Prevention and Research Institute of Texas (RP160283, RP17005)
- U.S. Department of Health & Human Services | NIH | National Cancer Institute (CA125123)
- U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (P30ES030285, P42ES0327725)
- John S. Dunn Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- human intestinal enteroids, organoids, CRISPR/Cas9 genome editing, lentiviral transduction, single-cell cloning, gene knockout, host restriction factors, innate immunity, intestinal diseases, ex vivo model
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Clone Cells, Cloning, Molecular, Humans, Lentivirus, RNA, Guide, Kinetoplastida, Gene Knockout Techniques, CRISPR-Cas Systems
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