Cell Culture stimulated T-cells: Cells were cultured at 37 °C, 5% CO2 in X-Vivo 15 (Lonza) supplemented with 5% human serum (Sigma-Aldrich, St. Louis, MO, USA), and 100 IU/mL human recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA) and 10ngmL-1 human recombinant IL-7 (BD Biosciences, San Jose, CA, USA). T cells were activated using immobilized anti-CD3 (clone OKT3, Tonbo Biosciences) and soluble anti-CD28 (clone CD28.2, Tonbo Biosciences) for 3 d before electroporation.
Nucleofection: stimulated T cells: Cells were resuspended in electroporation solution, mixed with the precomplexed RNP and electroporated using a 4D-Nucleofector (Lonza) using program EO-115. To assess homologous recombination, cells were transduced with rAAV after electroporation. CD34+: All Cas9 proteins (WT and mutants) were obtained from Integrated DNA Technologies. RNP complexes were made by mixing Cas9 with sgRNA at a molar ratio of 1:2.5 (Cas9:sgRNA) at 25 °C for 10 min before electroporation. CD34+ cells were electroporated with RNP at a concentration of 300 µ g mL-1 (unless stated otherwise). CD34+ cells were
resuspended in either P3 buffer (Lonza) or buffer 1M67 and electroporated using the Lonza 4D Nucleofector (with program DZ100). Cells were plated at 250,000– 500,000 cells/mL in cytokine-supplemented medium, and then rAAV6 was delivered onto cells immediately (within 15 min) upon plating after electroporation
ranging vector genomes per cell from 50,000–100,000 as titered by qPCR or 5,000–10,000 as titered by droplet digital PCR (ddPCR). HEK 293 screening has been performed in the 96Well Shuttle system.