Human CAR T cell generation

For aITGB2 CAR T cells, the cells were then additionally nucleofected with ribonuclease complex of ITGB2 sgRNA and Cas9 using a P3 Primary Cell 4D-Nucleofector X kit S (Lonza, V4XP-3032) and 4D-Nucleofector (Lonza) with its built-in program EO-115.

Mouse CAR T cell generation

Mouse T cells were cultured and activated for 24?h using Dynabeads Mouse T-Expander CD3/CD28 (Gibco, 11452D) and magnetically removed thereafter. T cells (2?×?10^6 ) were nucleofected with Cas9 ribonuclease complex (RNP; Lonza, V4SP-3096) using a 4D-Nucleofector 96-well unit (Lonza, AAF-1003S) and Lonza program code DN-100 with 60?pmol of Cas9 protein (QB3 MacroLab) and 120?pmol of sgRNA (UCCCUCCUCUAGAACUUCAC; Synthego) preincubated at 37?°C for 10–15 min. The culture medium was then added to the cells and incubated (37?°C, 5% CO2) for 1?h.

Generation of ITGB2-knockout cells

Knockout cell lines or primary T cells were generated using in vitro nucleofection of Cas9 ribonuclease protein complex. Briefly, 2?µl of each sgRNA (100?µM; Synthego Corporation) and recombinant Cas9 protein (40?µM; QB3 MacroLab, University of California, Berkeley) was incubated at 37?°C for 15?min to generate ribonuclease complex, which was then nucleofected using a 4D-Nucleofector (Lonza) with the built-in program DS-137 for cell lines (using Lonza V4XC-2032) and EO-115 for primary T cells (using Lonza V4XP-3032). The sgRNAs used in this study were obtained from the Brunello library (Supplementary Table 3).