About

The data presented in this web portal are from pooled, image-based CRISPR knockout screens in HeLa cells. First, a fixed-cell primary screen targeted 5072 fitness-conferring genes, with phenotypes measured using widefield fluorescence microscopy of stains for DNA (DAPI), tubulin (antibody), gamma-H2AX (antibody), and actin (phalloidin). A secondary screen collected live-cell image phenotypes of cells expressing mCherry-tagged Histone H2B for 239 of these gene targets. The HeLa cells used in these screens have doxycycline-inducible Cas9 expression for precise control of the timing between knockout and phenotype measurement for these fitness-conferring genes. For the primary screen, cells were fixed 78 hours after Cas9 expression induction. In the live-cell secondary screen, we tracked cells for 24 hours (10 minute time resolution) for two separate time courses: 48 hours -> 71 hours post-Cas9 induction (referred to as “Day 3”) and 72 hours-> 96 hours post-Cas9 induction (“Day 4”). The pooled knockouts were demultiplexed in situ and matched to the individual cell phenotype images using the pooled optical pooled screening approach (see Feldman, et al. ).

This project is a collaboration between the Cheeseman Lab at the Whitehead Institute and the Blainey Lab at the Broad Institute, with special thanks to Andy Nutter-Upham and Scott McCallum at the Whitehead Institute for their generous work in developing this web portal.

Cell image montages
The cells displayed in each montage were randomly selected from all acquired images for each sgRNA. For the primary screen, up to 100 cells are displayed for each sgRNA (median of 1,454 acquired in total for each sgRNA). Separate secondary screen montages are included for the day 3 and day 4 time courses, with up to 150 mitotic events included for each sgRNA (median of 290 acquired in total across sgRNAs and timepoints). These timelapse montages were computationally aligned such that all cells enter mitosis at approximately the same frame, and are in sorted order by mitotic event duration. Timestamps denote time relative to approximate mitotic entry, and the 10 frames before mitotic entry and after mitotic exit are included in the montage if available (i.e., are within the acquired time course).

Fixed-cell primary screen features
The fixed-cell measurements included in this data portal are a small subset of the 1084 total phenotype features extracted from each cell image, chosen based on their combined ability to discriminate between functional phenotypes and for their relatively simple interpretation. The values reported here are gene-level summary scores for each feature. These scores are computed by taking the median of values across individual cells expressing the same sgRNA, and then taking the median of these values among sgRNAs targeting the same gene. Feature values in this portal are also normalized, meaning the values are standardized by the median and median absolute deviation of cells expressing non-targeting sgRNAs within the same well, prior to summary scoring. Reported FDR q-values were computed by comparing summary scores to null distributions of corresponding bootstrapped summary scores from cells expressing non-targeting guides to generate raw p-values (bootstrap sample size matching the cell and sgRNA sample size of each gene target, 100,000 bootstrap repetitions each for cell and sgRNA levels), and then applying the Benjamini-Hochberg procedure to obtain FDR bounds.

The features are defined in hierarchical form:

  1. Cell cycle phase: either “interphase” or “mitotic”
  2. Compartment: either “nucleus” or “cell”
  3. Feature type: “correlation”, “shape”, or the name of the image channel for intensity-based measurements
  4. Feature name: explained in the following table

Feature Name Feature Type Analogous CellProfiler feature Explanation Interpretation of normalized values
mean intensity Intensity_MeanIntensity The mean of channel intensity values within the segmented compartment. Negative values: decreased mean channel intensity compared to cells expressing non-targeting sgRNAs
Positive values: increased mean channel intensity compared to cells expressing non-targeting sgRNAs
integrated intensity Intensity_IntegratedIntensity The sum of channel intensity values within the segmented compartment. Negative values: decreased integrated channel intensity compared to cells expressing non-targeting sgRNAs
Positive values: increased integrated channel intensity compared to cells expressing non-targeting sgRNAs
mass_displacement intensity Intensity_MassDisplacement The euclidean distance between the segmented compartment's geometric centroid and the channel intensity weighted centroid (e.g., center of gravity of the channel intensity values). Negative values: channel intensity is more evenly distributed across the compartment compared to cells expressing non-targeting sgRNAs
Positive values: channel intensity is more unevenly distributed across the compartment compared to cells expressing non-targeting sgRNAs
mean_edge intensity Intensity_MeanIntensityEdge The mean of channel intensity values within pixels bordering the edge of the segmented compartment. Edge is defined with connectivity of 2 (includes pixels diagonally-adjacent to the edge). Negative values: decreased mean channel intensity of edge pixels compared to cells expressing non-targeting sgRNAs
Positive values: increased mean channel intensity of edge pixels compared to cells expressing non-targeting sgRNAs
std_edge intensity Intensity_StdIntensityEdge The standard deviation of channel intensity values within pixels bordering the edge of the segmented compartment. Edge is defined with connectivity of 2 (includes pixels diagonally-adjacent to the edge). Negative values: decreased variability of edge pixel channel intensity compared to cells expressing non-targeting sgRNAs
Positive values: increased variability of edge pixel channel intensity compared to cells expressing non-targeting sgRNAs
mean_frac_0 intensity RadialDistribution_MeanFrac_{channel}_1of4 First, the segmented compartment is divided into 4 concentric rings centered on the point farthest from the compartment edge; the width of each ring is 1/4th of the radius at the approximate angle of the compartment. This feature is the mean fractional intensity within the innermost ring; calculated as the fraction of the object's total integrated channel intensity that is within this ring, normalized by the relative area of the ring. Negative values: channel intensity is less concentrated at the center of the compartment compared to cells expressing non-targeting sgRNAs
Positive values: channel intensity is more concentrated at the center of the compartment compared to cells expressing non-targeting sgRNAs
mean_frac_3 intensity RadialDistribution_MeanFrac_{channel}_4of4 First, the segmented compartment is divided into 4 concentric rings centered on the point farthest from the compartment edge; the width of each ring is 1/4th of the radius at the approximate angle of the compartment. This feature is the mean fractional intensity within the outermost ring; calculated as the fraction of the object's total integrated channel intensity that is within this ring, normalized by the relative area of the ring. Negative values: channel intensity is less concentrated within the outermost ring of the compartment compared to cells expressing non-targeting sgRNAs
Positive values: channel intensity is more concentrated within the outermost ring of the compartment compared to cells expressing non-targeting sgRNAs
{channel1}{channel2} correlation Correlation_Correlation Pearson correlation coefficient between pixel-level intensity values of two channels within the segmented compartment. Negative values: pixel intensities of the two channels within the compartment are less correlated than for cells expressing non-targeting sgRNAs
Positive values: pixel intensities of the two channels within the compartment are more correlated than for cells expressing non-targeting sgRNAs
area shape AreaShape_Area 2D surface area of the segmented compartment Negative values: decreased compartment area compared to cells expressing non-targeting sgRNAs
Positive values: increased compartment area compared to cells expressing non-targeting sgRNAs
eccentricity shape AreaShape_Eccentricity Eccentricity of the ellipse that has the same second-moments as the segmented compartment. The eccentricity is the ratio of the focal distance of the ellipse (distance between focal points) over the major axis length. The value is in the interval [0, 1). Eccentricity of 0 is equivalent to a circle, eccentricity approaching 1 is a line segment. Negative values: compartment shape is more circular than in cells expressing non-targeting sgRNAs
Positive values: compartment shape is more elongated than in cells expressing non-targeting sgRNAs
form_factor shape AreaShape_FormFactor The ratio of the segmented compartment's area to the area of a circle with the same perimeter as the compartment; equivalent to the isoperimetric quotient. The value is in the interval (0, 1]. Form factor of 1 is equivalent to a circle. Negative values: compartment has a smaller perimeter relative to its area compared to cells expressing non-targeting sgRNAs
Positive values: compartment has a larger perimeter relative to its area compared to cells expressing non-targeting sgRNAs
solidity shape AreaShape_Solidity Ratio of the segmented compartment's area to the area of the convex hull of the compartment. The value is in the interval (0, 1]. Solidity of 1 is a shape that is fully convex. Negative values: compartment is less convex compared to cells expressing non-targeting sgRNAs
Positive values: compartment is more convex compared to cells expressing non-targeting sgRNAs
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