Cas9prot

For mRNA transfection, we used an mRNA encoding enhanced green fluorescent protein (eGFP) with ARCA cap modifications (Apexbio Technologies, Fisher Scientific #50-199-8310). For Cas9 RNP complex transfection, 24 µL of 60 µM crRNA (Supplementary Table 2) was hybridized with 12 µL of 120 µM atto550-tagged Atr-R™ tracrRNA (Integrated DNA technologies) in Tris-buffered saline by warming to 80 °C for 5 min, then cooling to 4 °C for 15 min. The hybridized RNAs were then mixed with 25 µL of 30 µM (5 mg/mL) recombinant SpCas9 protein (Sigma-Aldrich #Cas9PROT) for 30 min at 4 °C, resulting in a molar ratio of 2:1:1 Cas9:crRNA:tracrRNA. T cell receptor (TCR) expression was quantified by FITC anti-human TCR α/β antibody (BioLegend #306706, clone IP26) and imaging flow cytometry. For each condition, about 100,000 cells were incubated in 100 µL of PBS with 0.1% bovine serum albumin (BSA, Sigma Aldrich #A9418) and 5 µL of antibody (i.e., 1:20 dilution) on ice for 30 min, then washed twice in chilled PBS with BSA. Viability, eGFP expression, and TCR immunocytochemistry were assessed by flow cytometry following the same gating strategy as prior studies with FITC–dextran, as described above and shown in Supplementary Fig. 25.