CD4+ T cell Isolation and Culture
CD4+ T cells were isolated from LRS chambers obtained from healthy donors at the Stanford Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll density gradient followed by CD4+ cell enrichment through negative selection using magnetic separation with LS columns (Miltenyi) and cryopreservation in Bambanker cell freezing media (Wako Chemicals). T cells were thawed and cultured at 106 cells/mL in X-VIVO15 with gentamicin (Lonza), 5% human serum (Corning) and 100 IU mL–1 human IL-2 (Peprotech) with (HIV-optimized culture) or without (Standard culture) 40 ng/mL IL-7 and 10 ng/mL IL-12. At thaw, CD4+ T cells were activated with anti-CD3/anti-CD28 paramagnetic beads for standard culture conditions (Dynabeads Human T Cell Activator, Gibco) or on culture plates coated with Ultra-LEAF TM Purified anti-human CD3 (Biolegend #317326, clone OKT3, lot B329667) for HIV-optimized culture. Cells were cultured at 37°C with 5% CO2 and ambient O2, and replated at optimum cell density every 2-3 days with fresh complete media. For editing experiments, cells were activated in culture for three days prior to nucleofection, and activation beads were removed before electroporation where relevant.

CCR5 knockout
sgRNAs were acquired from TriLink Biotechnologies with 20-O-methyl-30-phosphorothioate modifications at the three terminal nucleotides of both ends and purified using HPLC. Guides sequences used for editing CCR5 were GCAGCATAGTGAGCCCAGAA (internal guide) or TGACATCAATTATTATACAT (start site guide). High-fidelity Cas9 protein was purchased from Aldeveron (SpyFi Cas9). RNPs were generated by precomplexing purified sgRNAs with HiFi SpCas9 protein at a molar ratio of 2.5:1 for 15-30 minutes at room temperature. RNPs were electroporated at a Cas9 concentration of 300 µg per mL using a 4D Nucleofector (Lonza) in 16-cuvette strips. 250,000 HSPCs or 106 activated T cells were nucleofected in P3 solution with program DZ100 or EO-115, respectively, with or without RNP and rAAV6 as noted.