primary isolated CD4+ T cells (by ficoll separation and negative Easy sep enrichment kit) were stimulated with CD3/CD28 for 48h in the presence of IL-2. Used Cas9 protein with two NLS signals to transfect in solution P3 and program EH-115 Details: Cas9 RNPs were prepared fresh for each experiment. crRNA and tracrRNA were first mixed 1:1 and incubated 30 min at 37°C to generate 40 µM crRNA: tracrRNA duplexes. An equal volume of 40 µM Cas9-NLS was slowly added to the crRNA:tracrRNA and incubated for 15 min at 37°C to generate 20 µM Cas9 RNPs. For each reaction, around 300,000 stimulated T cells were pelleted and resuspended in 20 µL P3 buffer (Lonza). 3 µL of 20 µM Cas9 RNP mix was added directly to these cells and the entire volume transferred to the 96-well reaction cuvette. For double-editing reactions, 3 µL of each Cas9 RNP was added to 20 µL cells. Cells were electroporated using program EH-115 on the Amaxa 4D-Nucleofector (Lonza). 80µL pre-warmed, complete RPMI was added to each well, and the cells were allowed to recover for 30 min at 37°C. Cells were then re-stimulated on plates coated overnight with 10 µg/mL anti-CD3 (UCHT1, Tonbo Biosciences) and 10 µg/mL anti-CD28 (CD28.2, Tonbo Biosciences) for 24 hr prior to infection.