CD34+ HSPCs. Mobilized peripheral blood (mPB) and bone marow (BM) CD34+ HSPCs cells were purchased from AllCells.

Editing of all CD34+ HSC and T-cells was carried out using a ribonucleic protein (RNP) system at a molar ratio of either 1:2.5 or 1:5 (Cas9: sgRNA), unless otherwise stated. Nucleofection was performed in P3 nucleofection solution (Lonza) and Lonza Nucleofector 4d (program DZ-100 for CD34+ and EO-115 for T-cells). Cells were plated at a concentration of 1.0 × 105–2.5 × 105 cells/ml. and transduced with the AAV6 donor at an multiplicity of infection (MOI) of 200,000 vg/µl within 15 min of nucleofection.

T-cell purification. Primary human T cells were obtained from healthy male donors from Stanford University School of Medicine Blood Center after informed consent was obtained and purified by Ficoll density gradient centrifugation followed by red blood cell lysis in ammonium chloride solution and magnetic negative selection using a Pan T-cell isolation kit (Miltenyi Biotec, San Diego, CA, USA) according to manufacturer’s instructions.
Cells were cultured at 37 °C, 20% O2 and 5% CO2 in X-Vivo 15 (Lonza, Walkersville, MD, USA) supplemented with 5% human serum and 100 IU/ml human recombinant IL-2  and 10 ng/ml human recombinant IL-7. Cells were stimulated with immobilized anti-CD3  and with soluble anti-CD28 (CD28.2, Tonbo Biosciences) for three days prior to electroporation.

U-2OS: sgRNAs were generated by cloning annealed oligos containing the IL2RG target sequence into pX330 (Gift from Feng Zhang, Addgene #42230)56. In all, 200,000 U2OS cells (ATCC #HTB-96) were nucleofected with 1 µg of pX330 Cas9 and gRNA plasmid and 100 pmol dsODN using SE cell line nucleofection solution and the CA-138 program on a Lonza 4D-nucleofector. The nucleofected cells were seeded in 500 µl of McCoy’s 5a Medium Modified (ATCC) in a 24-well plate.