human T cell: 1 million primary human T cells were nucleofected with 10 µg of the indicated synthetic CCR5 sgRNAs and either 15 µg Cas9 mRNA or 1 µg Cas9-encoding plasmid.

Stimulated T cells: were nucleofected as above, but with 15 µg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs

 CD34+ HSPCs from mobilized peripheral blood were maintained in X-VIVO 15 (Lonza) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3-Ligand (100 ng/ml), IL-6 (100 ng/ml) and StemRegenin1 (0.75 mM).

CD34+ HSPCs were nucleofected using the Lonza 4D-Nucleofector (program EO-100) and the P3 Primary Cell 4D-Nucleofector Kit (V4XP-3024).
Nucleofection conditions: 100 µl nucleofection solution, 5 × 105 cells, 10 µg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 µg Cas9 mRNA or 1 µg Cas9 plasmid. See also 2nd pdf with supplementary information

1 million stimulated T cells or mobilized human peripheral blood CD34+ HSPCs were nucleofected with 15 µg Cas9 mRNA and 10 µg of the indicated synthetic CCR5 sgRNAs. When used in combination the amount of each sgRNA was 5 µg. Before nucleofection, T cells were activated for 3 d with immobilized anti- CD3 antibodies (clone: OKT3, eBioscience, San Diego, CA, USA) and soluble anti-CD28 antibodies (clone: CD28.2, eBioscience).

For non-activated CD3+ T cells, cells were Nucleofected immediately after isolation. T cells were Nucleofected using the Lonza Nucleofector 2b (program U-014) and the Human T Cell Nucleofector Kit (VPA-1002, Lonza).
Nucleofection conditions: 100 µl Nucleofection solution, 106 cells, 10 to 20 µg chemically modified sgRNA,15 to 30 µg Cas9 (or 15 µg eGFP mRNA, TriLink BioTechnologies, San Diego,CA, USA), 1 µg sgRNA/Cas9-encoding plasmid.