Fig. 1: CRISPR/Cas9 drop-out screening identifies genetic vulnerabilities in CUX1-deficient cells. | Nature Communications

Fig. 1: CRISPR/Cas9 drop-out screening identifies genetic vulnerabilities in CUX1-deficient cells.

From: Cut-like homeobox 1 (CUX1) tumor suppressor gene haploinsufficiency induces apoptosis evasion to sustain myeloid leukemia

Fig. 1

a Scheme of CRISPR/Cas9 drop-out screen to identify selective vulnerabilities in CUX1−/− cells. WT, wild-type. b Immunoblot showing loss of CUX1 protein expression in three CUX1−/− clones compared with wild-type (WT). HDAC1 and β-Actin were used as loading controls. The experiment was done twice with similar results. c Partial sequence of CUX1 exon 18 from CUX1−/− clones 1D, 1E and 2D showing wild-type genomic and amino acid (aa) sequences (top) and clone-specific mutated sequences (bottom); inv, inversion. d Distribution of residual scores as a measure of gene essentiality in CUX1−/− cells compared with wild-type cells. Positions of CFLAR, AKT1 and AKT2 genes are highlighted. Target genes with higher residual scores exhibit higher degree of potential synthetic lethality in CUX1−/− than in wild-type cells. e Functional annotation of dependency genes in CUX1−/− cells identified by CRISPR/Cas9 screening. PUD, proteasomal ubiquitin-dependent. P values were not adjusted for multiple comparisons. Apoptosis pathway is highlighted in red. f Competitive sgRNA assay (left) showing preferential loss of CFLAR sgRNA-targeted cells in CUX1−/− U937 cells compared with wild-type. The results from CUX1−/− U937 cells were normalized to wild-type cells at each time point. Day 5 was designated a starting value of 100% and subsequent values were calculated relative to day 5. Immunoblot (right) confirming depletion of target protein by CFLAR-targeting sgRNA compared with control sgRNA (Con) in wild-type and CUX1−/− cells. The experiment was done three times. g Immunoblot showing expression of CFLAR-V5 in CUX1−/−:Cas9 cells (left) and rescue of lethality following CFLAR-targeting sgRNA infection in CFLAR-V5-expressing cells (right). Empty vector was used as control (vector). Results were normalized to day 4 in each case. Statistically significant comparisons shown are between empty vector-expressing groups. The experiment was done three times. h Percentage of Annexin-V+ cells at day 5 following CFLAR-targeting sgRNA infection compared with control sgRNA (Con) in CUX1−/− cells. The experiment was done three times. i Immunoblot of indicated proteins at day 5 following CFLAR-targeting sgRNA infection compared with control sgRNA (Con) in CUX1−/− cells. The experiment was done twice with similar results. CUX1−/− clone 1D cell line was used in f–i. One-way ANOVA with Dunnett’s test for multiple comparisons and repeated measures (f, g), Two-tailed, unpaired t-test (h).

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