Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: A) Workflow for image-based screening of dynein cargo localisation. B, C) Representative images and quantification of immunostained unmodified U-2 OS cells following CRISPR/Cas9-mediated editing of LIS1 (B) or DYNC1H1 (C). NTC, non-targeting control; Hoescht, 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 relocalisation assay. Only one motor complex is depicted per peroxisome for simplicity. 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 relocalisation assay in high-throughput format. Scatter plot and corresponding violin plots (median, bold line; first/third quartile, dashed lines) of number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots. Data points represent robust-Z (rZ) normalisation (central reference = NTC treated with rapamycin; value increases with cargo dispersion) with mean aggregation at well level (minimum of 100 wells analysed from 3 × 384-well plates). rZ′ values show assay window between NTC with rapamycin and crLIS1 with rapamycin. G) Representative images and quantification of early endosome (EEA1) dispersion in unmodified U-2 OS cells after indicated treatments. Bar graph shows ratio between EEA1 spot number at the perinuclear region vs the peripheral region (lower values indicate increased dispersion). Data points represent mean aggregation at the well level (minimum of 100 cells analysed per well, four wells analysed per condition). Error bars, S.D.. ***p<0.001 (one-way ANOVA with Dunnett’s multiple comparison vs NTC + DMSO). Scale bar, 20 μm.

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: CRISPR, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY, High Throughput Screening Assay

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: A) Workflow for phenotypic profiling using images collected from the secondary screen. B) UMAP plot for phenotypes of genes selected from the primary screen. Clusters of genes with well-defined functions are highlighted. C) Phenotypic feature heatmap of the ‘dynein-dynactin’ gene cluster. Features are grouped in the x-axis according to the marker (see zoomable ‘Supplementary phenotypic heatmap’ file and Supplementary table 7 for names of individual features). Genes encoding dynein and dynactin components, as well as the associated proteins BICD2 and LIS1, are labelled in different shades of blue, whereas FHF component genes are shown in magenta. Novel genes in the cluster are labelled in grey. The scales of rZ values (central reference = NTC) were adjusted based on min and max values of individual features. ‘Cytoplasm’ refers to features associated with the background Hoescht staining in the cytoplasm.

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: Marker, Staining

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: Heatmap displaying localisation ratio of dynein cargoes at the perinuclear vs peripheral region of cells treated with the indicated crRNAs. GFP-BICD2N-FRB and PTS-RFP-FKBP were evaluated in U-2 OS PEX cells treated with rapamycin, whereas other markers were evaluated in unmodified, untreated U-2 OS cells. crRNAs were synthesised based on the Vienna Bioactivity CRISPR score (labelled ‘(V)’), except for LIS1 and DYNC1H1 crRNAs from the initial Discovery set (labelled ‘(D)’), which were used as additional positive controls. Bold labelling indicates crRNAs that target novel constituents of the ‘dynein-dynactin’ cluster previously generated by unsupervised profiling. Colour scales of individual features were adjusted based on their min and max values. Categories of affected cargoes were manually annotated based on statistically significant effects (see Supplementary figure 12). Data represent relative change of the mean aggregated at well level compared to NTC from a minimum of three independent experiments (minimum of 100 cells analysed per well, four wells analysed per condition).

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: CRISPR, Generated

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: A) Schematic of SUGP1 domain structure. NLS, nuclear localisation signal. B) Representative images of SUGP1 intensity and GFP-BIC2N-FRB and PTS-RFP-FKBP localisation in U-2 OS PEX cells treated with crSUGP1 #1. Scale bar, 25 μm. C) Scatter plot of mRNA abundance for crSUGP1 #1-edited vs NTC U-2 OS cells (mean log 2 normalised values from three independently performed experiments). mRNAs meeting the threshold for inclusion (minimum absolute log 2 fold change ≥ 0.5; FDR ≤0.05) are labelled in blue, except SUGP1, DYNC1I2 and LIS1 , which are labelled in yellow. Inset table shows non-logarithmic values for SUGP1, DYNC1I2 and LIS1 . See Supplementary table 8 for full results. D) Quantification of endogenous DYNC1I2 and LIS1 protein signal (assessed by immunofluorescence) in unmodified U-2 OS treated with NTC, crSUGP1 #1 or #2 and transfected with a control (iRFP670) or crRNA-resistant SUGP1-V5 expression plasmid. E) Representative images and quantification (perinuclear vs peripheral localisation ratio) of GFP-BICD2N-FRB and PTS-RFP-FKBP localisation in U-2 OS PEX cells treated with NTC, crSUGP1 #1 or #2 and transfected with a control (iRFP670), crRNA-resistant SUGP1-V5, or LIS1-FLAG expression plasmid. Scale bar, 25 μm. In D and E, cells were transfected with crRNA 96 h before fixation, and with expression plasmid 48 h after crRNA transfection. Data points are aggregated means of independent experiments, with a minimum of 100 transfected cells analysed per condition. Error bars signify S.D.. * p <0.05, ** p <0.01, *** p <0.001 (two-way ANOVA with Tukey’s multiple comparison; colours of asterisks indicate comparison group).

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation