ReviewNature protocols2022
Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.
Review in Nature protocols, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers.
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Who cites it
40 citing papers in PubMed, 57 citations in OpenAlex.
- Immune responses in healthy adults elicited by a bivalent norovirus vaccine candidate composed of GI.4 and GII.4 VLPs without adjuvant.Frontiers in immunology · 2023Trial
- Synthesis of RARα-PROTAC and its delivery viaMaterials today. Bio · 2026Article
- Tumor-Induced PDPNInternational journal of molecular sciences · 2026Article
- Inducing physiological polarity and performing gene editing using CRISPR-Cas9 in human trophoblast organoids.Nature protocols · 2026Review
- Organoids: From Bench to Bedside Applications.MedComm · 2026Review
- Reprogramming gut microenvironment for the treatment of acute severe ulcerative colitis via a synergistic therapy of necroptosis blockade and organoid transplantation.Nature communications · 2026Article
- microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2.Molecular therapy. Nucleic acids · 2026Article
- Organoids Gone Viral: A Comprehensive Review on Human Organoid Models to Study Viral Pathogenesis.Viruses · 2026Review
- Overcoming host restrictions to enable continuous passaging of GII.3 human norovirus in human intestinal enteroids.Science advances · 2026Article
- Intestinal epithelial TLR5 signaling promotes barrier-supportive macrophages.Science immunology · 2026Article
- Nanoscale synthetic biology with innovative medicinal applications.Fundamental research · 2025Review
- CRISPR-based functional genomics tools in vertebrate models.Experimental & molecular medicine · 2025Review
- SAMD9 senses cytosolic double-stranded nucleic acids in epithelial and mesenchymal cells to induce antiviral immunity.Nature communications · 2025Article
- How to Study the Mechanobiology of Intestinal Epithelial Organoids? A Review of Culture Supports, Imaging Techniques, and Analysis Methods.Biology of the cell · 2025Review
- PRMT5 Maintains Homeostasis of the Intestinal Epithelium by Modulating Cell Proliferation and Survival.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2025Article
- Loss of mucin 2 and MHC II molecules causes rare resistance to murine RV infection.Journal of virology · 2025Article
- Organoid development and applications in gynecological cancers: the new stage of tumor treatment.Journal of nanobiotechnology · 2025Review
- Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches.Frontiers in immunology · 2025Article
- Zbtb7b defines a compensatory mechanism in MASLD-related HCC progression by suppressing H19-mediated hepatic lipid deposition.Physiological reports · 2024Article
- Advancing cancer research through organoid technology.Journal of translational medicine · 2024Review
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 2 countries.
Funding
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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.