Examination of surrounding genomic features of eGSHs and association with functional activity. (A) 400-kbp region centered on the GSH peak for GSHs 1 to 6 and GSHs 7, 12, 20, and 30 are shown. The red flash mark indicates the position of the gRNA targeted at each GSH. Indicated are Refseq coding genes in dark blue, noncoding genes in light blue, pseudogenes in orange, and eGSH region in light green, ATAC-seq peaks in activated cells obtained from our data (donor 2 is used as a representative) in red, and in resting cells, obtained from data in the study by Corces et al,⁵⁸ in dark green. The signal intensity for both sets of data are scaled to the same range for all panels. (B) The 263 kb genomic region around GSH6 is shown encompassing the 2 closest genes on either side of GSH6. Genes are illustrated in blue and the corresponding polymerase chain reaction (PCR) amplicons used for quantitative reverse transcriptase polymerase chain reaction are shown in red along with their names below. The red flash symbol indicates the gRNA cut site. (C) RNA expression of genes around GSH6 represented as ΔCt in comparison to 18s rRNA. Two primer pairs were used for ZNF746 and 1 each for KRBA1 and ZNF767P. Data are shown as mean ± SD of n = 5 to 9 technical replicates from RNA of CAR T cells at day 0 and day 7 after stimulation on CD19+ aAPCs (CAR protein expression of the same cells is shown in Figure 2E). Dotted line indicates minimum nontemplate control ΔCt value, that is, no expression (taken from CAR expression values in untransduced cells); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; 2-way ANOVA with Dunnett multiple comparison test.