Figure 1. Domain-focused CRISPR/Cas9 screening identifies core TFs essential for MCL proliferation and survival.

(A) Experimental schematic for the CRISPR/Cas9 screen and the competition-based GFP dropout proliferation assay. (B) Scatterplot analysis of TF dependencies in CCMCL1 cells (y axis) versus HEL cells (x axis) ranked by the average sgRNA log₂ fold change (log₂FC) of each gene in the pooled CRISPR screen. (C) Heatmap depicts log₂FC of sgRNA abundance of selected genes (averaging each independent sgRNA targeting a gene). (D) Competition-based proliferation assays to validate the results from the pooled screen. Experiments were conducted by transduction of Cas9-expressing JEKO1 cells with indicated lentivirus sgRNAs that coexpress a GFP reporter. Plotted is the percentage of GFP-positive cells (normalized to the day 3 measurement) at the indicated time points during culturing. sgRNAs targeting ROSA and MYC are included as a nontargeting negative control and a positive control, respectively. (E–H) Verification of on-target effects of sgRNAs against IRF4 (E), PAX5 (F), EBF1 (G), and FOXO1 (H). Competition-based proliferation assays in CCMCL1 cells expressing control or 3× FLAG–tagged and CRISPR-resistant synonymous IRF4 (F-IRF4r^#1), PAX5 (F-PAX5r^#1), EBF1 (F-EBF1r^#1), and FOXO1 (F-FOXO1r^#1) mutants. The indicated CRISPR-resistant synonymous mutants were designed specifically for sgIRF4#1, sgPAX5#1, sgEBF1#1, and sgFOXO1#1. (I) Violin plots of RNA expression levels in transcripts per million (TPM) of indicated TFs in patient MCL cells (n = 37). RNA-Seq data were reanalyzed from GSE141336 of Zhao et al. (38). (J and K) Immunoblot analysis of patient MCL cells (J) and patient-derived xenografts (PDX) (K). (D–H) Data represent mean ± SEM (n = 3). Results are representative of 2 independent experiments. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.001, ***P < 0.0005, ****P < 0.0001.