Successful scale-up of CRISPR-Cas9–mediated NR3C1 deletion in VSTs. (A) Different VST numbers (3 × 10⁶, 25 × 10⁶, and 100 × 10⁶) were electroporated in the presence of Cas9 and gRNA using the Lonza 4D nucleofector and the NR3C1 KO efficiency in Cas9 (control) or NR3C1 KO VSTs was determined using PCR (A; n = 3) and western blot analysis (B; n = 3). β-actin was used as loading control in panel B. The percentages (%) of NR3C1 KO efficiency (A) and GR protein loss (B) after CRISPR-Cas9 gene editing are shown under each figure. (C) Representative FACS plots showing the percentage of apoptotic cells (annexin V⁺) and alive or dead cells (live/dead stain) in control Cas9 and NR3C1 KO VST cells at the different cell dose levels of 3 × 10⁶, 25 × 10⁶, or 100 × 10⁶ cells per electroporation treated with or without Dexa (200 μM) for 72 hours (n = 3). (D) Graph summarizing the absolute cell number between control Cas9 (solid lines) and NR3C1 KO VST cells (dotted lines) at the different cell dose levels of 3 × 10⁶ (green), 25 × 10⁶ (blue), or 100 × 10⁶ (red) cells per electroporation for 72 hours (n = 3). Bars represent mean values with SD. (E-F) Bar graphs showing the percentage of IFN-γ, TNF-α, or IL-2 production by 100 × 10⁶ control Cas9 (green), 100 × 10⁶NR3C1 KO (blue), or 100 × 10⁶NR3C1 KO plus dexamethasone (Dexa 200 μM; red) VSTs in response to 6-hour stimulation with the relevant viral PepMix in the CD8⁺ T-cell (E) and CD4⁺ T-cell (F) compartments (n = 3). The functional analysis of the Cas9+Dexa group was not performed due to the absence of viable cells resulting from the lymphocytotoxic effect of steroids. The bars represent mean values with SD.