ArticleJournal of visualized experiments : JoVE2017
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.
Article in Journal of visualized experiments : JoVE, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 17 citations in OpenAlex.
- TCA cycle rewiring underpins histone acetylation sourcing and cell-fate transitions during exit from naive pluripotency.Cell stem cell · 2026Article
- Universal conditional knockout approach to multiple species, from fish to human induced pluripotent stem cells.Nucleic acids research · 2026Article
- Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids.Molecular and cellular biochemistry · 2026Article
- WNK kinase regulates plasma membrane levels of the WNT inhibitor RNF43: A WNK-RNF43 circuit regulates WNT signal suppression.bioRxiv : the preprint server for biology · 2025Article
- Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2.Science advances · 2023Article
- p57Cell stem cell · 2022Article
- Efficient gene editing in a medaka (Journal of Zhejiang University. Science. B · 2022Article
- USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.EMBO reports · 2021Article
- Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line.International journal of molecular sciences · 2020Article
- Human Microphysiological Models of Intestinal Tissue and Gut Microbiome.Frontiers in bioengineering and biotechnology · 2020Review
- Advancing insights into stem cell niche complexities with next-generation technologies.Current opinion in cell biology · 2018Review
- A novel cloning strategy for one-step assembly of multiplex CRISPR vectors.Scientific reports · 2018Article
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 3 countries.
Funding
Abstract
CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.
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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.