Multiplex editing and non-viral knock-in using primary T cells

Human primary CD4+ and CD8+ T cells were activated for 48–72 h prior to nucleofection. T cells were debeaded according to the manufacturer’s protocol and counted by Trypan blue exclusion on a NucleoCounter NC-200 (ChemoMetec, Denmark). Prior to nucleofection, ribonucleoprotein (RNP) mixtures with spCas9 (Aldevron, Fargo, ND), single-guide (sg) RNAs (Synthego, Redwood City, CA), and poly-L-glutamic acid (PGA, Sigma-Aldrich, St. Louis, MI) (100 mg/mL) at volumetric ratios of gRNA (1): PGA (0.8): Cas9 (1) were used. To prepare RNP, 10 mg/mL Cas9 (stock concentration: 62 µM) was diluted in Cas9 storage buffer (Aldevron) to 40 µM and then mixed with 40 pmol of spCas9 (1 µL/1e6 cells), 250 pmol of TRAC sgRNA (0.375 µL/1e6 cells), B2M sgRNA (1 µL/1e6 cells) and PDCD1 sgRNA (1.13 µL/1e6 cells), and PGA (2 µL/1e6 cells). RNP was then incubated at 37°C for 15–30 min. For CAR T-cell production, 1–2 µg of the dsDNA HDR template was added to the RNP for 5 min at room temperature. T cells were centrifuged at 400 g for 5 min, re-suspended in 18 uL of the Lonza P3 buffer, and added to the RNP/DNA mixture. T cells were nucleofected using a Lonza 4D-Nucleofector (Lonza, Walkersville, MD), with programs EO-115 for multiplex knockout and EH-115 for non-viral CAR knock-in. Edited T cells were recovered at 37°C and 5% CO2 for 5–10 min in cuvettes with 80 uL of RPMI media supplemented with IL-7 and IL-15 or IL-2. Cells were then added to a 48-well plate and expanded in RPMI media supplemented with IL-7 and IL-15 or IL-2 for 7 days