HEK293T cells were cultured in DMEM with 10% heat-inactivated FBS, 2 mM GlutaMax (ThermoFisher, 35050–061), 50 U/ml penicillin-streptomycin (ThermoFisher, 15070063) at 37 °C and 5% CO2 and seeded at 3 × 10^5 cells/mL 24 hrs before nucleofection. To prepare the PE2 protein-pegRNA/gRNA complex (RNP), approximately 600 pmol of the PE2 protein was incubated with 800 pmol of the pegRNA and 267 pmol of the nicking guide RNA (Agilent Technologies, Supplementary Table 6) in 320 mM KCl in 8.6 µl total volume for 15 min at room temperature. HEK293T cells (2 × 10^5) were nucleofected with 2 µl of the RNP using SF Cell Line 4D-Nucleofector™ X Kit S (Lonza, V4XC-2032) with the ED-130 program.
For PE experiments, the T cells (1 × 10^6 cells/mL) were cultured in RPMI 1640 medium containing 2 mM GlutaMAX (Thermo Scientific, 72400–120), 10% FBS, 1% penicillin-streptomycin (100 IU/ml; Thermo Scientific, 15140122) and 20 IU/mL recombinant human interleukin-2 (Peprotech, 200–02) for 24 hrs and activated with 1.5% phytohemagglutinin M (Thermo Scientific, 10576015). Approximately 48 hrs later, 5 × 10^5 cells were electroporated with RNP (prepared as above) via the EH-115 program using the P3 primary cell nucleofector kit (Lonza, V4XP-3024) and a 4D-Nucleofector. Electroporation conditions for primary T cells were previously optimized using the pmaxGFP vector (Lonza) and flow cytometry analysis (Supplementary Data 1). The cells were cultured at 1 × 10^6 cells/mL for approximately 72 hrs post electroporation.