Fig. 3: Cargo specificity of validated hits.

A–E RPE-1 cells stably expressing Cas9 nuclease were transfected in 96 wells plates with individual synthetic sgRNAs, and after 3 days, were subjected to 24 h uptake of the following cargoes: A dextran-Oregon Green 488, B EGF-A488, C transferrin-A488, D Tau oligomers-Cy3, and E α-syn-PFFs-A633 as controls of sgRNAs’ effects. Cells were then fixed, and nuclei were stained with Hoechst 33342. Imaging was done using a CX7 high-content microscope and quantification with the HCS Studio Cell Analysis software. The mean total fluorescence intensity per cell was measured for each cargo. Images are shown in Suppl. Fig. 4A. Only Tau oligomers showed important changes in uptake upon invalidation of some hits, especially EXT1, NDST1, and TM9SF2 which are known to affect heparan sulfate expression, and VPS35 which was previously reported to increase the uptake of proteinaceous aggregates. Intriguingly, C3orf58 and SLC39A9 did not significantly decrease Tau oligomers-uptake. n = 3–4 independent experiments. For the AAVS1 negative control, two separate sgRNAs were used. Graph bars represent mean ±s.d. Statistical test: one-way ANOVA: *p < 0.05, ***p < 0.001, ****p < 0.0001.