T cell nucleofection: T cells were activated using Dynabeads CD3/CD28 CTS (Gibco, Life Technologies) at a 3:1 bead-to-cell ratio. 24 hrs after bead activation, T cells were transduced with lentivirus at an MOI of 3. 48 hrs after stimulation, beads were removed and T cells were spun down at 300 xg, washed with PBS once and resuspended at a concentration of 5e6 cells/100 µL in P3 Solution with Supplement (Lonza). Primary T cells were electroporated using the Lonza 4D-Nucleofector Core/X Unit and the P3 Primary Cell 4-D Kit (Lonza). For CRISPR/Cas knockouts, the ribonucleoprotein (RNP) complex was formed by incubating 20 µg of Spy Fi Cas9 (Aldevron) with 10 µg of sgRNA (Table S3) (IDT) for 15 mins at room temperature (RT). The RNP complex and 100 µL of resuspended cells were combined and electroporated in a cuvette. For base-editing, 10 µg mRNA (Trilink, N1-Methylpseudouridine, CleanCap) encoding the base-editor (ABE8e) were mixed with 10 µg sgRNA (IDT) and combined with 100 µL of resuspended cells. Pulse code EO-115 were used for primary T cells. After electroporation, the cells were recovered in CTS Optimizer at 2e6 cells/mL at 37°C for 24 hrs. Cells were counted daily using the Multisizer 4 Coulter Counter (Beckman Coulter). T cells were grown for 8–10 days in Optimizer media containing 5 ng/ml of IL-7 and IL-15 and maintained at 1e6 cells/ml prior to cryopreservation. T cells were thawed and rested at 37°C for 16 hours before experiments.
Primary human CD34+ cells isolation and electroporation: G-CSF mobilized peripheral blood samples were obtained healthy volunteers enrolled on an IRB approved protocol (University of Pennsylvania IRB protocol # 832307). Human subjects were deidentified and thus age and sex information are not available. CD34+ selection was performed using the CD34 Microbead Kit (Miltenyi Biotec, 130–046-702), and purity was confirmed by flow cytometry to be >95%. Cells were cryopreserved in FBS with 10% DMSO until use. Prior to all experiments, CD34+ cells were thawed and cultured in StemSpan SFEM (Stem Cell Technologies, 09650) supplemented with human cytokines (SCF 100 ng/µl, Flt3 ligand 100 ng/µl, TPO 50 ng/µl, IL-6 50 ng/µl, all purchased from Peprotech) for 24 hrs. Nucleofection was performed as described for T cells using pulse code DZ-100. Cells were recovered at 2 x 10^6 cells/mL in StemSpan SFEM (Stem Cell Technologies, 09650) supplemented with human cytokines (SCF 100 ng/µl, Flt3 ligand 100 ng/µl, TPO 50 ng/µl, IL-6 50 ng/µl, all purchased from Peprotech) for 24 hrs at 37°C.
Primary human monocyte base editing: Human monocytes were obtained from PBMCs of healthy donors through the Human Immunology Core at the University of Pennsylvania. Monocytes were cultured in RPMI 1640 + 5% human serum, 1% penicillin/streptomycin (50 IU/ml), 1% Glutamax) (Gibco, Life Technologies) supplemented with 50 ng/mL of MCSF (Peprotech) at 37°C in 5% CO2. After 24 hrs in culture, monocytes were nucleofected with ABE8e mRNA and sgRNA as described for T cells using pulse code CM-137. Cells were recovered at 2 x 10^6 cells/mL in RPMI 1640 + 5% human serum, 1% penicillin/ streptomycin (50 IU/ml), 1% Glutamax) (Gibco, Life Technologies) supplemented with 50 ng/mL of M-CSF (Peprotech) and cultured for 5 days prior to experimental use to allow for sufficient protein turnover.