P3 primary cell nucleofector solution with supplement 1

CRISPR-Cas9–mediated gene knockout in primary T cells was performed with some modifications to a previous protocol (Seki and Rutz, 2018 (link); Oh et al., 2019 (link)). Briefly, naïve T cells from KRN transgenic mice were purified by negative isolation and assessed for counts and purity. Each sgRNA (Synthego, sequences listed below) was incubated with Alt-R S.p. Cas9 Nuclease V3 (Integrated DNA Technologies) for 10 min at room temperature to form a separate RNP complex, then combined into a single tube for each nucleofection condition. Cells were resuspended in P3 Primary Cell Nucleofector Solution with Supplement 1 (Lonza) plus Alt-R Cas9 Electroporation Enhancer (Integrated DNA Technologies) and immediately aliquoted to the tubes with combined RNP complexes. Mixed cells and sgRNA-Cas9 complexes were transferred to nucleocuvette wells (Lonza) and pulsed with the DN100 program on a Lonza 4D-Nucleofector. Electroporated cells were resuspended and washed in complete media, then assessed again for viable cell counts. KRN T cells were resuspended in sterile PBS prior to intravenous transfer (2 × 10⁵ cells) into B6.I-A^b/g7 recipients. CRISPR sgRNA sequences used are given in Table 2.

Hematopoietic stem and progenitor cells were counted after two days and centrifuged at 300 g for 10 minutes. T-cells were counted after 4–6 days in culture. Supernatant was removed and cells were resuspended in P3 Primary Cell Nucleofector^® Solution with Supplement 1 (Lonza, V4XP-3032) at 5–15 million cell/mL. Cell mixture was transferred to Lonza strip at 20 μL per well and electroporated using program DZ100 with solution setting P3. 80 μL of StemSpan^™ SFEM II supplemented with StemSpan^™ CD34+ Expansion Supplement (10X) (StemCell Technologies, Catalog #09720) was added to each well. AAV was added after electroporation, if necessary, at 10⁴ vg/cell. HSPCs were transferred to 24-well non-treated plates at a density of 100,000 cells/mL and incubated at 37°C for 2–3 days. T-cells were cultured at densities of 500,000–1,000,000 cells/mL.