Fig. 3: Cas9-RNP and AAV6-mediated targeting of human primary stem cells using i53 peptide.

a CD34⁺ HSPCs were electroporated with Cas9-RNP and AAV6 with or without i53 peptide targeting different genes (HBB, CCR5, IL2RG, HBA1). HDR-mediated outcomes were assessed by ddPCR 2 days post-electroporation. Data from n = 7 independent biological replicates for HBB targeting without i53, n = 5 HBB + i53, n = 3 for CCR5 without i53, n = 5 CCR5 + i53, n = 4 for IL2RG and HBA1. Mean ± SD depicted for all experiments. *P < 0.05 and **P < 0.005, respectively, by two-tailed paired t-tests (P = 0.002 for HBB; P = 0.0039 for IL2RG; P = 0.0271 for HBA1). Source data are provided as a Source Data file. b Indel frequencies were determined by ddPCR, ICE, or TIDE analysis 2 days post-electroporation. Data from n = 7 independent biological replicates for HBB targeting without i53, n = 5 HBB + i53, n = 3 for CCR5 without i53, n = 5 CCR5 + i53, n = 4 for IL2RG. Mean ± SD depicted. **P < 0.005 (P = 0.0063 for HBB) by two-tailed paired t-test. Source data are provided as a Source Data file. c Representative FACS plots of biallelic targeting using HBB-mCherry and HBB-GFP encoding AAV donors. Representative FACS plot from n = 3 biologically independent experiments. d Various cell types (Airway basal cells, T cells, and MSCs) were gene-edited with or without i53 peptide, HDR frequencies in airway stem cells were determined by ICE or TIDER analyses. HDR in MSCs were determined by the read out of GFP expressing cells via flow cytometry. In airway basal cells, CFTR locus was targeted to correct and restore ΔF508¹¹; in T cells, TRAC locus was targeted to integrate a CD19-specific chimeric antigen receptor inframe³⁷; in MSCs, HBB locus was targeted to integrate a GFP reporter transgene³⁸. All analyses were conducted 2–3 days post-electroporation unless noted otherwise. Data from n = 6 independent biological replicates for airway stem cells, n = 3 for T cells, and n = 3 for MSCs. Mean ± SD depicted.