Assay development for arrayed CRISPR/Cas9 screening. (A) Workflow for image-based screening of dynein cargo localization. (B and C) Representative images and quantification of immunostained, unmodified U-2 OS cells following CRISPR/Cas9-mediated editing of LIS1 (B) or DYNC1H1 (C). Hoechst, DNA stain; cr, crRNA. Violin plots show fluorescence intensity values at the single-cell level (minimum of 100 cells from at least four wells for each group; median, bold line; first/third quartile, dashed lines). ***P < 0.001 (two-tailed Mann–Whitney-test). Scale bar, 200 µm. (D) Illustration of inducible peroxisome relocalization assay. (E) Representative images of U-2 OS PEX cells stained for microtubules (α-Tubulin) and DNA (Hoechst) after the indicated treatments. Cells were treated with either DMSO (vehicle), rapamycin alone, or rapamycin with nocodozole (Noc) for 2.5 h before fixation. Scale bar, 20 µm. (F) Validation of inducible peroxisome relocalization assay in high-throughput format. Scatter plot and corresponding violin plots (median, bold line; first/third quartile, dashed lines) of the number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots. Data points represent rZ normalization (central reference = NTC treated with rapamycin; value increases with cargo dispersion) with mean per cell values aggregated at well level (minimum of 100 wells analyzed from 3 × 384-well plates). rZ′ values show an assay window between NTC with rapamycin and crLIS1 with rapamycin. Shift on plot for NTC + DMSO versus NTC + Rapamycin conditions is due to the combination of concentration of GFP-BICD2N-FRB on peroxisomes and perinuclear clustering of these structures. (G) Representative images and quantification of early endosome (EEA1) dispersion in unmodified U-2 OS cells after indicated treatments. The bar graph shows the ratio between EEA1 spot number in the perinuclear region versus the peripheral region (lower values indicate increased dispersion). Data points represent mean per cell values aggregated at well level (minimum of 100 cells analyzed per well; four wells analyzed per condition). Error bars, SD. ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison versus NTC + DMSO). Scale bar, 20 µm.
Figure 1.

Assay development for arrayed CRISPR/Cas9 screening. (A) Workflow for image-based screening of dynein cargo localization. (B and C) Representative images and quantification of immunostained, unmodified U-2 OS cells following CRISPR/Cas9-mediated editing of LIS1 (B) or DYNC1H1 (C). Hoechst, DNA stain; cr, crRNA. Violin plots show fluorescence intensity values at the single-cell level (minimum of 100 cells from at least four wells for each group; median, bold line; first/third quartile, dashed lines). ***P < 0.001 (two-tailed Mann–Whitney-test). Scale bar, 200 µm. (D) Illustration of inducible peroxisome relocalization assay. (E) Representative images of U-2 OS PEX cells stained for microtubules (α-Tubulin) and DNA (Hoechst) after the indicated treatments. Cells were treated with either DMSO (vehicle), rapamycin alone, or rapamycin with nocodozole (Noc) for 2.5 h before fixation. Scale bar, 20 µm. (F) Validation of inducible peroxisome relocalization assay in high-throughput format. Scatter plot and corresponding violin plots (median, bold line; first/third quartile, dashed lines) of the number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots. Data points represent rZ normalization (central reference = NTC treated with rapamycin; value increases with cargo dispersion) with mean per cell values aggregated at well level (minimum of 100 wells analyzed from 3 × 384-well plates). rZ′ values show an assay window between NTC with rapamycin and crLIS1 with rapamycin. Shift on plot for NTC + DMSO versus NTC + Rapamycin conditions is due to the combination of concentration of GFP-BICD2N-FRB on peroxisomes and perinuclear clustering of these structures. (G) Representative images and quantification of early endosome (EEA1) dispersion in unmodified U-2 OS cells after indicated treatments. The bar graph shows the ratio between EEA1 spot number in the perinuclear region versus the peripheral region (lower values indicate increased dispersion). Data points represent mean per cell values aggregated at well level (minimum of 100 cells analyzed per well; four wells analyzed per condition). Error bars, SD. ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison versus NTC + DMSO). Scale bar, 20 µm.

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