P3 nucleofection buffer

Frozen PBMCs were thawed and activated. Transductions were performed 24 hours post-activation using lentivirus (Alstem) at MOI 10. 24 hours post-transduction, primary T cells were transfected with CRISPR-Cas9:sgRNA complexes. Briefly, cells were collected and washed with PBS before resuspending in supplemented P3 nucleofection buffer (Lonza). A quantity of 20 pmol of Cas9 (Synthego) was combined with 130 pmol B2M-targeting sgRNA (Synthego) and incubated in P3 nucleofection buffer before addition to cells. Twenty microliters of the cell and ribonucleoprotein (RNP) mixture was transferred to a 16-well Nucleocuvette Strip and electroporated with the 4D nucleofector using the stimulated T cell program (EO-115). The cells were recovered in 100 µL of prewarmed media, X-VIVO 15 (Lonza) supplemented with 5% HIA human AB serum and 300 IU/mL IL-2. PBMCs were cultured and expanded in LymphoONE Tcell Expansion Medium (Takara Bio) supplemented with 1% HIA human AB serum and 300 IU/mL IL-2 for 6 days. Post expansion, positively transduced primary T cells were enriched using anti-PE microbeads (Miltenyi) according to manufacturer’s instructions against Protein L-biotin:streptavidin-PE using LS column. Cytotoxicity of B2M KO primary T cells was then assessed exactly as described previously.

24-48 hours post-transduction primary T cells were transfected with CRISPR-Cas9:sgRNA complexes. Briefly, cells were collected and washes with PBS before resuspending in supplemented P3 nucleofection buffer (Lonza). A quantity of 20 pmol of Cas9 (Synthego) was combined with 130 pmol B2M-targeting sgRNA (IDT) and incubated in P3 nucleofection buffer before addition to cells. Twenty microliters of the cell and ribonucleoprotein (RNP) mixture was transferred to a 16-well Nucleocuvette Strip and electroporated with the 4D nucleofector using the stimulated T cell program (EO-115). The cells were recovered and expanded in X-vivo media (Lonza) supplemented with 1% human serum (GeminiBio) and IL-2 (300 IU/ml). B2M KO was checked by flow cytometry after staining with anti-HLA-I antibody (W6/32).