Two days after T-cell activation, cells were electroporated to enable site-specific knock-in using
Cas9 RNPs. All electroporation experiments were performed on the 4D-NucleofectorTM System X Unit (Lonza, Basel, Switzerland) using the EH-115 program. RNPs were pre-complexed at a sgRNA:Cas9 ratio of 4.5:1, prepared by adding 3 µL of 60 µM sgRNA (Synthego Menlo Park, CA, USA) to 1 µL of 40 µM Cas 9 (QB3 Macrolab, University of California, Berkeley, CA, USA), and incubated for 10 min at room temperature (RT). Complexed RNPs were used right away or frozen fr later use. Sequences for all sgRNAs can be found in Table S2. T cells (0.6 x 106 or 1.0 x 106) were re-suspended in 17 µL P3 buffer including supplement 1 (Lonza). Subsequently, 4 µL of RNP complex was added together withthe dsDNA template donor (2 µg/3 µL unless stated otherwise) and incubated for 10 min at room temp. The RNP and dsDNA mix were added to the cell mixture and 23 µL was added to the transfection vessel and electroporated. After electroporation, 80 µL of recovery media (RPMI (GE Healthcare Life Sciences, Marlborough, MA, USA) including 20% FBS (GE Healthcare Life Sciences, Marlborough, MA, USA), 1% GlutaMAX-I (Invitrogen, Carlsbad, CA, USA), IL-7 at 10 ng/mL, and IL-15 at 5 ng/mL) was added to the electroporation vessel. The cells were rested for 30 min at 37 C and 5% CO2 before being transferred into a 48-well, tissue culture plate with 650 µL of recovery media. Two to three days after electroporation, the FBS concentration was reduced to 10% in the T-cell culture media with cytokines. T cell s were split every 3–4 days and fresh IL-7 and IL-15 cytokines were added.