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. 2024 Apr 29;12:RP90683. doi: 10.7554/eLife.90683

Figure 4. A two-tier loss-of-function screen for modulators of the response to WIN site inhibitors (WINi).

(A) Two-tier screen design. In the first tier, Cas9-expressing MV4;11 cells were transduced with a genome-wide sgRNA library and treated with 2 µM C6 until a resistant cell population emerged. sgRNA representation in the pretreatment population was compared to the post-treatment population (n = 2). In the second tier, cells were transduced with a custom library of distinct sgRNAs targeting non-pan-essential ‘hits’ from the first tier, cultured in the presence of DMSO, C6, or C16, and sgRNA representation in C6/C16-treated cultures compared to that from DMSO-treated cultures (n = 2). (B) Volcano plot, showing gene-level changes in sgRNA representation from the first tier (orange indicates false discovery rate [FDR] < 0.05). Datapoints corresponding to TP53, RPL22, and CDKN2A are indicated. See Figure 4—source data 1 for full output of the tier 1 screen. (C) Comparison of gene-level changes in sgRNA representation in C6- and C16-treated populations in the second tier screen, each compared to DMSO-treated populations (red indicates FDR < 0.05; black indicates non-targeting control sgRNAs). See Figure 4—source data 2 for full output of the tier 2 screen. (D) Top: overlap of genes from the tier 2 screen with enriched (left) or depleted (right) sgRNAs in C6- and C16-treated MV4;11 populations, compared to the DMSO control. Bottom: overlap of genes with enriched (left) or depleted (right) sgRNAs in the first versus second tiers of the screen. ‘Tier 1’ contains only those genes targeted in the tier 2 screen. ‘Tier 2’ contains the intersection of genes with altered sgRNAs in both the C6 and C16 treatments. (E) Ranked heatmap, representing the mean gene-level Log2 fold change (FC) of sgRNAs from the C6 and C16 treatments in the tier 2 screen, as well as gene enrichment analysis outputs. Note that ‘Signal transduction by p53 class mediator’ is a GO:BP term (orange); ‘p53’ assignments (yellow) were added by manual curation.

Figure 4—source data 1. Output of the tier 1 screen.
Figure 4—source data 2. Output of the tier 2 screen.

© 2024, BioRender Inc

Figure 4A was created using BioRender, and is published under a CC BY-NC-ND license. Further reproductions must adhere to the terms of this license.

Figure 4.

Figure 4—figure supplement 1. Genome-wide CRISPR screen identifies genes that influence response to C6/C16.

Figure 4—figure supplement 1.

(A) Tier 1 screen: daily cell counts of MV4;11 Cas9 and MV4;11 Cas9+GeCKOv2 (Library) populations treated with either DMSO or 2 µM C6. The two replicates of this screen are shown separately. (B) Normalized counts of each sgRNA (x-axis) in the GeCKOv.2 library targeting TP53 in the initial transduced cells (red; not visible on this scale) and the C6-treated population (blue). Data represents means of replicates; * indicates false discovery rate (FDR) < 0.05. (C) As in (B) but for sgRNAs targeting CDKN2A. (D) Schematic of the CDKN2A gene locus with indicated sites complementary to tier 1 and tier 2 screen sgRNAs. Red sgRNAs increase in representation in CRISPR screens. (E) miRNet 2.0 (Chang and Xia, 2023) analysis of the 27 miRNAs enriched in the tier 1 screen produced a single significant hit corresponding to the KEGG p53 signaling pathway. The miRNAs are represented as blue boxes and target genes as red circles; the connections between them are indicated. (F) As in (B) but for sgRNAs targeting RPL22. (G) Volcano plots, showing gene-level changes from the tier 2 screen in sgRNA representation in C6- (left) and C16- (right) treated populations compared to DMSO control cultures (orange indicates FDR < 0.05). (H) Graph depicting gene-level Log2 FC and FDR values for genes that were flagged as C6- (squares) or C16- (circles) specific in the tier 2 screen. (I) GO enrichment analysis of the 57 C6/C16 common genes emerging from tier 2 of the screen. Italics represent the number of genes in each category.