CRISPR-Cas9–mediated deletion of the gene coding for the GR in primary human VSTs. (A) Schematic summary of the protocol for CRISPR-Cas9–mediated KO of NR3C1 in primary human VSTs. PBMCs were stimulated with virus-specific PepMix from CMV, BKV, and adenovirus (in combination) in the presence of 10 ng/mL IL-7, 50 IU/mL IL-2, and 10 ng/mL IL-15. On days 7 to 10 of in vitro expansion, primary human VSTs were nucleofected with control Cas9 alone (Cas9 control) or Cas9 preloaded with gRNA targeting the exon 2 of the NR3C1 gene, which encodes for the GR protein. KO efficiency and functional assays were performed at day +14 post initial PBMC isolation. (B) Schematic representation of CRISPR-Cas9–mediated NR3C1 KO using 2 short guide crRNAs targeting the exon 2 of NR3C1 gene. (C-D) The NR3C1 KO efficiency of VSTs after electroporation with Cas9 alone (control), Cas9 complexed with 1 crRNA (crRNA 1 or crRNA 2), or Cas9 complexed with the combination of 2 crRNAs (crRNA 1 plus crRNA 2) using a WT Cas9 was determined by PCR analysis at day 3 (C) or western blot analysis at day 7 (D) after electroporation. The percentages (%) of NR3C1 KO efficiency (C) and GR protein loss (D) after CRISPR-Cas9 gene editing are shown under each figure. PAM, protospacer adjacent motif.