Fig. 1: Genome-wide clustered regularly interspaced short palindromic repeats (GW CRISPR) knockout (KO) screening of CD19 chimeric antigen receptor (CAR) T cells identified CUL5 as a candidate gene to enhance CAR T-cell survival.

a Outlines of the GW CRISPR KO screening of CD19 CAR T cells. After Day 10 of the culture, GW CRISPR KO-CD19 CAR T cells were repeatedly stimulated with γ-irradiated Raji cells. DNAs from CAR T cells at Days 10 and 40 were compared using next-generation sequencing. Stim, stimulation; EP, electroporation; tdx, transduction. b GFOLD score data and calculation. Genes with GFOLD greater than 2 were ranked. Individual ranking scores were added up from top to bottom (n = 3). c GFOLD score summary of three replicated data from three donors. The right panel depicts the expansion of the dotted area. d Secondary CRISPR KO screening of CD19 CAR T cells. Each of the six candidate genes was knocked out using two different sgRNAs. Data from experiments on three different donors are acquired and presented as the mean and standard error of the mean. e, f CUL5 KO using two different sgRNAs and its efficiency. The immunoblots show CUL5 expression in sgRNA-transduced CD19 CAR T cells (e). CUL5 to β-actin expression ratio in sgRNA-transduced CD19 CAR T cells (f). (n = 3, different donors) (g, h) Green fluorescent protein (GFP) positivity in different types of CUL5 KO-CAR T cells over time. CD19 CAR T cells (g) and CD37 CAR T cells (h) were stimulated by γ-irradiated tumor cells. Raji cells were used for Ag stimulation in both CD19 and CD37 CAR T cells. GFP-positive cells were sgRNA-transduced cells. (n = 4 for Ctrl sgRNA and CUL5 sgRNA#4690, n = 3 for CUL5 sgRNA#8377, different donors in g, n = 3, different donors in (h) Data were expressed as mean ± SEM. One-way ANOVA was used for comparing Ctrl and sgCUL5 (f). Two-way ANOVA with Tukey’s multiple comparisons test (with adjustment) was used for (g and h). Source data are provided as a Source Data file.