eGSHs that satisfy criteria 1 to 7 are efficiently targeted by CRISPR/Cas9 to express a functional CAR. (A) GSH atlas safety criteria (blue box) and ATAC-seq atlas criteria (green box). The plot represents ranked average maximum signal intensities of ATAC-seq peaks associated with 379 eGSHs (without pseudogenes atlas) across 7 cell replicates. The top 6 highest-intensity GSH peaks are highlighted. Bars show mean ± standard deviation (SD) of n = 7 cell replicates. (B) Volcano plot depicting the 379 eGSHs centered on the GSH peak with a 5-kb region on each side of the peak in 7 cell samples. Peaks are arranged in decreasing order of their maximum (peak summit) signal intensities. Color indicates the value of signal intensities. The eGSH coverage column depicts the region that falls under GSH criteria 1 to 6 in yellow and the region that falls outside the criteria in blue. (C) Cleavage efficiency at top 6 candidate eGSHs. Above, a zoomed-in view of an example candidate eGSH peak spanning 1865 bps and the 4 gRNAs (indicated in red flash symbols) tested for the eGSH at the summit of the peak. Below, CRISPR/Cas9 cleavage efficiencies of 4 independent gRNAs (each independent symbol) at the 6 top eGSHs. (D) (top) rAAV6 gene cassette for targeted integration with the 1928ζ-1xx CAR construct (EF1α promoter + intron, flanked by 300-bp homology arms). (bottom) Experimental schema for CAR integration and preparation of CAR T cells for in vitro cytotoxicity assays. (E) Flow plots of CAR expression from T cells transduced with GSH-CARs at day 3 after transduction before CAR purification. (F) Cytotoxicity assay for the CD19-CAR targeted at GSH 1, 2, and 3 and the TRAC locus (Firefly luciferase-expressing NALM6 as target cells). Data are shown as mean ± SD of 3 technical replicates from the same donor. RPM, reads per million.