HSPC xenotransplantation in NBSGW mice

NOD.Cg-Kit^W-41JTyr ⁺Prkdc^scidIl2rg^tm1Wjl/ThomJ (NBSGW) mice were housed in a pathogen-free facility. Control or edited mobilized healthy donor or non-mobilized SCD CD34⁺ cells (0.4 to 1.2 × 10⁶ cells per mouse) were transplanted into nonirradiated NBSGW male and female mice of 5 to 6 weeks of age via retro-orbital sinus injection. NBSGW male and female mice transplanted with non-mobilized SCD CD34⁺ cells were conditioned with busulfan (Sigma, St Louis, MO, USA) injected intraperitoneally (10 mg/kg body weight/day) 24 h, 48 h and 72 h before transplantation. Neomycin and acid water were added in the water bottle. 16 to 20 weeks after transplantation, NBSGW primary recipients were sacrificed. Cells were harvested from bone marrow, thymus, spleen, and blood, stained with antibodies against murine and human surface markers [murine CD45 (1/50 mCD45-VioBlue), Miltenyi Biotec; human CD45 (1/50 hCD45-APCvio770), Miltenyi Biotec; human CD3 (1/50 CD3-APC), Miltenyi Biotec; human CD14 (1/50 CD14-PE-Cy7), BD Biosciences; human CD15 (1/50 CD15-PE), Miltenyi Biotec; human CD19 (1/100 CD19-BV510); human CD235a (1/50 CD235a-PE), BD Biosciences] and analyzed by flow cytometry using the MACSQuant analyzer (Miltenyi Biotec) and the FlowJo software (BD Biosciences). The gating strategy used to assess chimerism and lineage-specific markers is shown in Supplementary Fig. ²². Human bone marrow CD45⁺ cells were sorted by immunomagnetic selection with AutoMACS (Miltenyi Biotec) after immunostaining with the CD45 MicroBead Kit (Miltenyi Biotec). All experiments and procedures were performed in compliance with the French Ministry of Agriculture’s regulations on animal experiments and were approved by the regional Animal Care and Use Committee (APAFIS#2019061312202425_v4). Mice were housed in a temperature (20–22 °C) and humidity (40–50%)-controlled environment with 12 h/12 h light/dark cycle and fed ad libitum with a standard diet.