A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.

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.

One-step generation of conditional and reversible gene knockouts.

Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.

Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids.

Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction.

A video protocol of retroviral infection in primary intestinal organoid culture.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these 'mini-guts', also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium.

Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.

Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors.

Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.

Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists.

Encapsulated cargo internalized by fusogenic liposomes partially overlaps the endoplasmic reticulum.

Few endocytosed ligands, including bacterial toxins and simian virus 40 (SV40) have been shown to reach the endoplasmic reticulum (ER) in mammalian cells. Using calcein and fluorescently labelled lactoferrin encapsulated in fusogenic liposomes we found that the cargo uses a microtubule-based pathway with ER delivery. Endocytic uptake of the lipid vesicles was cholesterol dependent in all cell lines tested, including the caveolin-1-deficient human hepatoma 7 cell line.