human T cells were stimulated with aCD3/ACD28 for 48h. Transfection of Cas9RNPs to disrupt PD-1 gene: Solution P3 and program EH-115: Primary human CD4+ or CD8+ T cells were thawed, rested for 24 hours in TCM followed by stimulation with plate-bound anti-CD3 (10 µg/mL; Tonbo Biosciences, clone: UCHT1) and soluble anti-CD28 (5 µg/mL; Tonbo Biosciences, clone: CD28.2) for 48 hours in T cell medium supplemented with IL-2 (30 U/mL). Electroporation was performed using the Amaxa P3 Primary Cell kit and 4D-Nucleofecter (Lonza). The recombinant S. pyogenes Cas9 used in this study expresses a C-terminal HA tag and two nuclear localization signal (NLS) peptides that facilitate transport across the nuclear membrane. The protein was expressed and purified as described37 and obtained from Macrolab, University of California, Berkeley. Cas9 was stored in 20 mM HEPES at pH 7.5, 150 mM KCl, 10% (v/v) glycerol, 1 mM TCEP at -80 °C. Cas9 RNPs were prepared fresh for each experiment as follows: chemically synthesized tracrRNA and crRNA (Dharmacon) targeting Pdcd1 exon 1 (PD-1 crRNA targeting sequence: 5'-CGACTGGCCAGGGCGCCTGT- 3'3) were re-suspended with 10 mM Tris-HCl pH 7.4 to generate 80 µM RNA stocks. crRNA and tracrRNA were mixed 1:1 and incubated 30 minutes at 37 °C to generate 40 µM crRNA:tracrRNA duplexes. An equal volume of 40 µM S. pyogenes Cas9-NLS was slowly added to the crRNA:tracrRNA and incubated for 15 minutes at 37 °C to generate 20 µM Cas9 RNPs. ~3 × 105 stimulated T cells were re-suspended in 20 µl P3 buffer and 3 µl of 20 µM Cas9 RNP mix added. Cells were nucleofected using program EH-115. 80 µl pre-warmed TCM was added to wells and cells allowed to recover 30 minutes at 37 °C. Cells were then seeded at 1 × 106 cells/mL in TCM containing Dynabeads Human T Activator anti-CD3/anti-CD28 (Invitrogen) at a bead:cell ratio of 1:1. Two-to-four hours later lentiviral supernatant was added to wells for 24–36 hours. Beads were removed 4–6 days following stimulation.