The genome-wide screen recovers known components of the dynein–dynactin machinery, as well as novel hits. (A) Plate layout for genome-wide screen. (B) Example of imaging data from the screen (maximum intensity projections of Z-stacks captured with a 20×/1.0 NA water objective). Scale bar, 50 µm. (C) Evaluation of editing efficiency across the screen using cell viability as a readout. Scatter plot and corresponding violin plot for controls (median, bold dashed line; first/third quartile, dashed lines) of the proportion of viable cells (gated based on nuclear morphology of NTC cells) per well after treatment with NTC, crLIS1, or crPLK1. Data points (color-coded as in A) are rZ normalized values (central reference = NTC). rZ′ value shows assay window between NTC and crPLK1. Library copies of crPLK1 and crLIS1 are labeled with “(L).” (D) Effects of arrayed library crRNAs on area and roundness of nuclei. Scatter plot and corresponding violin plots (median, bold dashed line; first/third quartile, dashed lines) of rZ normalized values (color coded as in A; central reference = NTC). Dashed lines represent ± 4*SD of NTC and crLIS1 (x-axis) or −3.5*SD of NTC and crLIS1 (y-axis), which were thresholds for hit calling. Genes previously shown to influence nuclear morphology are highlighted. (E and F) Example of endpoints used for hit selection from peroxisome (E) and early endosome (F) data. Scatter plot and corresponding violin plots (median, bold dashed line; first/third quartile, dashed lines) of normalized values (color-coded as in A) based on the neutral control (NTC, 0) and positive control (crLIS1, −100); rZ′ values show assay window between NTC and crLIS1 control wells. Dashed lines on the x- and y-axes represent, respectively, thresholds of −2*SD (E) or −3*SD (F) used for hit calling. In F, “EEA1 morphology” values were generated from a linear discriminant analysis weighted average of three endpoints quantifying the symmetry, intensity profile, and texture of EEA1 signal. “EEA1 localization ratio” is the ratio between the number of EEA1 spots in the perinuclear region versus the peripheral region. Core components of dynein and dynactin that met the threshold for hit calling are labeled with purple and teal text, respectively. Other categories of genes highlighted in the text are labeled in gold or orange.
Figure 2.

The genome-wide screen recovers known components of the dynein–dynactin machinery, as well as novel hits. (A) Plate layout for genome-wide screen. (B) Example of imaging data from the screen (maximum intensity projections of Z-stacks captured with a 20×/1.0 NA water objective). Scale bar, 50 µm. (C) Evaluation of editing efficiency across the screen using cell viability as a readout. Scatter plot and corresponding violin plot for controls (median, bold dashed line; first/third quartile, dashed lines) of the proportion of viable cells (gated based on nuclear morphology of NTC cells) per well after treatment with NTC, crLIS1, or crPLK1. Data points (color-coded as in A) are rZ normalized values (central reference = NTC). rZ′ value shows assay window between NTC and crPLK1. Library copies of crPLK1 and crLIS1 are labeled with “(L).” (D) Effects of arrayed library crRNAs on area and roundness of nuclei. Scatter plot and corresponding violin plots (median, bold dashed line; first/third quartile, dashed lines) of rZ normalized values (color coded as in A; central reference = NTC). Dashed lines represent ± 4*SD of NTC and crLIS1 (x-axis) or −3.5*SD of NTC and crLIS1 (y-axis), which were thresholds for hit calling. Genes previously shown to influence nuclear morphology are highlighted. (E and F) Example of endpoints used for hit selection from peroxisome (E) and early endosome (F) data. Scatter plot and corresponding violin plots (median, bold dashed line; first/third quartile, dashed lines) of normalized values (color-coded as in A) based on the neutral control (NTC, 0) and positive control (crLIS1, −100); rZ′ values show assay window between NTC and crLIS1 control wells. Dashed lines on the x- and y-axes represent, respectively, thresholds of −2*SD (E) or −3*SD (F) used for hit calling. In F, “EEA1 morphology” values were generated from a linear discriminant analysis weighted average of three endpoints quantifying the symmetry, intensity profile, and texture of EEA1 signal. “EEA1 localization ratio” is the ratio between the number of EEA1 spots in the perinuclear region versus the peripheral region. Core components of dynein and dynactin that met the threshold for hit calling are labeled with purple and teal text, respectively. Other categories of genes highlighted in the text are labeled in gold or orange.

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