Freshly purified primary T cells were activated for 3 days according to the procedure described above, and then transduced with lentiviral vectors harboring the CD19 CARs. Two days after transduction, CAR-T cells were electroporated with Cas9 protein (ThermoFisher Scientific) and the intended sgRNAs targeting the TCRa constant chain (TRAC), ß2-microglobulin (B2M) exon1, and PD-1 exon1 by 4D-Nucleofector System N (Lonza) using the P3 Primary Cell 4D-Nucleofector X Kit, V4XP-3024 (Lonza). Cas9:single-guide RNA ribonucleoproteins(Cas9 RNPs) were prepared immediately before experiments by incubating Cas9 protein with sgRNA at a 1:1 ratio at room temperature for 10 min. 3×10^6 cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS, ThermoFisher Scientific) by centrifuging at 200g for 5 minutes and resuspended in 100 µL transfection buffer containing Cas9 RNP and then transferred into the electroporation cuvette. Program EO-115 was selected for high efficiency. After electroporation, cells were resuspended in 2 mL pre-warmed T cell medium and transferred into a 12-well cell plate and incubated at 37 °C in 5% CO2. The transfection efficiency was evaluated 3 and 7 days after electroporation. Cell culture medium was half replaced by fresh complete medium every 2 ~ 3 days.