Fig. 6

CRISPR/Cas9 gene editing in blood–brain barrier organoids to investigate the mechanisms of transcytosis. a Representative confocal images of blood–brain barrier organoids assembled with hCMEC/D3 Cas9 knockout cells incubated with a human Brain Shuttle antibody for 4 h. The upper images show an overlay of human IgG signal (yellow) and nuclei labelled with DAPI (cyan). The intensity of the lower images was scaled to visualize the background intensity in the antibody channel (grey). Scale bar, 100 μm. b Quantification of IgG intensity within blood–brain barrier organoids. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles. Differences between the scrambled control and transferrin receptor or clathrin heavy-chain knockout organoids were statistically significant (*p < 0.05) whereas the difference between the scrambled control and caveolin-1 knockout organoids was not statistically significant (p = 0.609). Comparisons were evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons of ~ 50 organoids per condition in n = 3 independent experiments. c Representative confocal images of human Brain shuttle antibody distribution in control (upper panels) or transferrin receptor knock-out (lower panels) blood–brain barrier organoids treated as in a. Panels on the left show a low magnification image of an organoid. Scale bar, 100 μm. Panels on the right show a high magnification image of the boxed area. Arrowheads point to accumulation of Brain Shuttle signal in puncta and/or tubules. Scale bar, 10 μm