Figure S4.
Additional genome-wide screen endpoints. (A) Effects of arrayed library crRNAs on cell viability and comparison to results from previous cell viability studies. Scatter plot of library results and corresponding violin plot for controls (median, bold dashed line; first/third quartile, dashed lines; color code in key at bottom of figure), proportion of inviable cells (gated based on nuclear morphology of NTC cells). Data points represent normalized values based on the neutral (NTC, 0) and lethal editing (crPLK1, 100) controls. Dashed line on y-axis represents 2.5*SD of crLIS1, the threshold for calling lethal crRNAs. Library copies of crPLK1, crBICD2 and crLIS1 are labeled with “(L).” The genes targeted by the lethal crRNAs were cross-referenced with their gene essentiality categorization from the Cancer Dependency Map project (DepMap 22Q2 Public+Score; https://depmap.org/portal): “common essential genes” are classed as essential for growth and survival in ≥90% of cancer cell lines; “essential for U-2 OS” genes are those classed as not essential across multiple cell lines but essential in U-2 OS cells; “non-essential” genes are those not identified as essential in a panel of cell lines; “not screened” genes are those that are not represented in the DepMap screening dataset. Numbers in parentheses refer to the percentage of essential genes in our dataset found in each Depmap category. (B) Effects of arrayed library crRNAs on micronuclei incidence. Scatter plot of library results and corresponding violin plot of controls (median, bold dashed line; first/third quartile, dashed lines; color code in key at bottom of figure) for the number of micronuclei per cell (x-axis) and number of cells containing micronuclei (y-axis). Data points represent rZ normalization (central reference = NTC). Dashed lines on the x- and y-axes represent 2.5*SD of crLIS1, the threshold for calling crRNAs that cause a micronucleus phenotype. The library copy of crPLK1 is labeled with “(L).” Labeled genes were functionally enriched (FDR ≤ 0.005) for GO terms associated with regulation of chromosome segregation, including, “nuclear division,” “mitotic sister chromatid segregation,” and “nuclear chromosome segregation” (http://bioinformatics.sdstate.edu/go/). (C) Effects of arrayed library crRNAs on PTS-RFP-FKBP texture and number of PTS-RFP-FKBP and GFP-BICD2N-FRB spots. Scatterplot of library results and corresponding violin plots of controls (median, bold dashed line; first/third quartile, dashed lines; color code in key at bottom of figure) of normalized values based on the neutral control (NTC, 0) and crLIS1 (−100). Dashed lines on both axes represent ± 2.5*SD of NTC and arrayed library, the threshold for hit calling. Both endpoints were generated from a linear discriminant analysis; PTS-RFP-FKBP texture was generated from six textural features based on filtered images, while “GFP/RFP spots” were generated by two features (total number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots). Core components of the dynein complex and dynactin complex that met the threshold for hit calling for either endpoint are labeled in purple and teal text, respectively. Library copy of crLIS1 is labeled with “(L).” Known peroxisome biogenesis genes are labeled in blue text (note duplicate of the PEX12 crRNA pool in the library). crPAFAH1B2 and crDNM1L are labeled as examples of crRNA pools that affect PTS-RFP-FKBP texture in the opposite way to crRNAs targeting dynein–dynactin components.

Additional genome-wide screen endpoints. (A) Effects of arrayed library crRNAs on cell viability and comparison to results from previous cell viability studies. Scatter plot of library results and corresponding violin plot for controls (median, bold dashed line; first/third quartile, dashed lines; color code in key at bottom of figure), proportion of inviable cells (gated based on nuclear morphology of NTC cells). Data points represent normalized values based on the neutral (NTC, 0) and lethal editing (crPLK1, 100) controls. Dashed line on y-axis represents 2.5*SD of crLIS1, the threshold for calling lethal crRNAs. Library copies of crPLK1, crBICD2 and crLIS1 are labeled with “(L).” The genes targeted by the lethal crRNAs were cross-referenced with their gene essentiality categorization from the Cancer Dependency Map project (DepMap 22Q2 Public+Score; https://depmap.org/portal): “common essential genes” are classed as essential for growth and survival in ≥90% of cancer cell lines; “essential for U-2 OS” genes are those classed as not essential across multiple cell lines but essential in U-2 OS cells; “non-essential” genes are those not identified as essential in a panel of cell lines; “not screened” genes are those that are not represented in the DepMap screening dataset. Numbers in parentheses refer to the percentage of essential genes in our dataset found in each Depmap category. (B) Effects of arrayed library crRNAs on micronuclei incidence. Scatter plot of library results and corresponding violin plot of controls (median, bold dashed line; first/third quartile, dashed lines; color code in key at bottom of figure) for the number of micronuclei per cell (x-axis) and number of cells containing micronuclei (y-axis). Data points represent rZ normalization (central reference = NTC). Dashed lines on the x- and y-axes represent 2.5*SD of crLIS1, the threshold for calling crRNAs that cause a micronucleus phenotype. The library copy of crPLK1 is labeled with “(L).” Labeled genes were functionally enriched (FDR ≤ 0.005) for GO terms associated with regulation of chromosome segregation, including, “nuclear division,” “mitotic sister chromatid segregation,” and “nuclear chromosome segregation” (http://bioinformatics.sdstate.edu/go/). (C) Effects of arrayed library crRNAs on PTS-RFP-FKBP texture and number of PTS-RFP-FKBP and GFP-BICD2N-FRB spots. Scatterplot of library results and corresponding violin plots of controls (median, bold dashed line; first/third quartile, dashed lines; color code in key at bottom of figure) of normalized values based on the neutral control (NTC, 0) and crLIS1 (−100). Dashed lines on both axes represent ± 2.5*SD of NTC and arrayed library, the threshold for hit calling. Both endpoints were generated from a linear discriminant analysis; PTS-RFP-FKBP texture was generated from six textural features based on filtered images, while “GFP/RFP spots” were generated by two features (total number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots). Core components of the dynein complex and dynactin complex that met the threshold for hit calling for either endpoint are labeled in purple and teal text, respectively. Library copy of crLIS1 is labeled with “(L).” Known peroxisome biogenesis genes are labeled in blue text (note duplicate of the PEX12 crRNA pool in the library). crPAFAH1B2 and crDNM1L are labeled as examples of crRNA pools that affect PTS-RFP-FKBP texture in the opposite way to crRNAs targeting dynein–dynactin components.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal