Fig. 5: CRISPR/Cas9 and AAV6 mediated targeted integration of a promoter-less cassette allows transgene expression under the control of the endogenous CX3CR1 promoter in hHSPCs.

A Experimental scheme. After thawing, hHSPCs were kept in prestimulation media enriched with a cytokine cocktail for 2 days. Then, cells were nucleofected with sgRNAs delivered as RNP complexes with CRISPR/Cas9 and transduced with the AAV6 donors. Cells were then kept in culture and analyzed for NHEJ, HDR, FACS, and gene expression to measure the efficiency of editing at the CX3CR1 locus. B Representative FACS plots of the hHSPCs nucleofected with CRISPR/Cas9 RNP with the chosen sgRNAs and transduced with the AAV6. GFP expression is indicative of the activity of the promoter (promoterless constructs) or efficiency of targeted integration (AAVS1 safe harbor control). C Percentage of GFP assessed by FACS in hHSPCs edited with CRISPR/Cas9 + AAV6 vectors in the tested and control conditions. D Percentage of targeted alleles assessed by ddPCR in the edited hHSPCs in the tested and control conditions. E CX3CR1 gene expression in edited hHSPCs in the tested conditions, shown as fold on mock. F CX3CR1 protein expression assessed by FACS in edited hHSPCs in the tested conditions shown as fold on mock. C–F Individual replicates and mean values ± SD are shown, 3 independent experiments. Source data are provided as a Source Data file.