Supplementary Figure 1: Gating scheme for identifying and quantifying the number of HSPCs with targeted integration of a GFP reporter gene.

FACS plots show the gating strategy for identifying CD34⁺ HSPCs targeted with a GFP reporter gene. The figure shows data from cord blood-derived CD34⁺ HSPCs that were electroporated two days after isolation with Cas9 RNP targeting the HBB locus. Immediately after electroporation, cells were transduced at an MOI of 50,000 using an AAV6 donor vector carrying a UbC-GFP expression cassette. Four days after electroporation and transduction, cells were stained for CD34 (and propidium iodide to identify live cells) and then analyzed on a FACS Aria (BD). Cells are identified in a forward/side scatter plot (FCS-A and SSC-A) and single cells discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells are discriminated based on propidium iodide, and in the final GFP/CD34 plot, the targeted GFP+CD34+ cells are identified. Cell frequencies within gates are shown.