Cell culture
Jurkat cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 2mM L-glutamine, 100 U/ mL penicillin, and 100 mg/mL streptomycin. Peripheral blood mononuclear cells were isolated from de-identified buffy coats obtained from healthy adult donors from the Aarhus University Hospital Blood Bank by Ficoll-Plaque plus density gradient and from these, primary human T cells were purified by negative selection with the EasySep human T cell isolation kit (STEMCELL Technologies). The primary human T cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 5% human albumin serum (Merck) and 10 ng/mL IL-7 and IL-2 (Peprotech). The cells were activated for 3 days with Dynabeads human T-activator CD3/CD28 (Thermo Fisher Scientific) at a 1:1 cell-to-bead ratio.

Electroporation
All cells were electroporated using the 4D-nucleofector device from Lonza (X unit) in 20-µL-format Nucleocuvette strips. Cells were electroporated in the following electroporation buffers and programs. Jurkat cells: Opti-MEM (Thermo Fisher Scientific), CM138-P3; primary human T cells: solution 1M, EO115-P3.49 For CRISPRa and CRISPRi singleplex RNA-based delivery experiments, unless otherwise specified, cells were electroporated with 0.095 µg/mL mRNA + 0.05 mg/ µL of each of the sgRNAs. For orthogonal CRISPRa and CRISPRi experiments, cells were electroporated with 0.095 µg/µL dSpCas9-VPR mRNA along with 0.0125 µg/µL of each sgRNA for CD123 (#1–4) and NGFR (#1–4), and 0.095 µg/µL dSaCas9-KOX1 mRNA along with 0.0167 µg/µL of each sgRNA for CD5 (#1–3) and CD3E (#1–3). In primary human T cells for CRISPRa and CRISPRi experiments for trimodal engineering, at the optimized condition cells were electroporated with 0.095 mg/mL dSpCas9-VPR mRNA + 0.0125 µg/µL of each sgRNA for CD123 (#1–4) and 0.095 µg/µL dSaCas9-KOX1 + 0.05 µg/ µL of each sgRNA for CD5 (#1–3).

For gene editing of TRAC with nuclease-active Cas9 protein (Alt-R S.p. Cas9 Nuclease V3; IDT), Cas9 and sgRNAs were incubated for 15 min at room temperature and later stored at 4°C prior to electroporation. RNP complexes were mixed with cells resuspended in 1 M electroporation buffer.50 Cas9 protein and sgRNAs were at a final concentration of 0.320 µg/µL and 0.160 µg/µL, respectively.