Fig. 6

HSPC-pDCs are amenable to genetic modifications. a Graphical illustration of the protocol for CRISPR/Cas9-mediated gene editing using Cas9 RNP. CD34⁺ HSPCs were initially cultured in CD34⁺ HSPC medium at low density for 3 days before being electroporated with Cas9 RNP complexes. After a 3-day recovery phase, pDC differentiation was initiated. b Expansion of cells during differentiation from HSPCs into HSPC-pDCs. c Indel frequencies after the 3-day recovery phase quantified by TIDE analysis for cells targeted at MyD88, IFNAR1, and CCR5 (control). d Numbers of HSPC-pDCs at day 21 for gene-edited HSPC-pDCs. e Western blot analysis showing induction of pSTAT1 upon 3 h IFN-β stimulation (upper panel) and protein levels of MyD88 (lower panel) in HSPC-pDCs targeted at IFNAR1 and MyD88, respectively (detection Ab for STAT1: CST 9172). Indel frequencies at the respective targets are listed below the western blot. f, g Surface expression levels (MFI) of CD123 (f) and CD304 (g) in IFN-primed and unprimed HSPC-pDCs with gene editing at MyD88, IFNAR1, and CCR5. h Functional levels of type I IFN after stimulation with R837 (TLR7) or CpG-A (TLR9) in unprimed and IFN-primed HSPC-pDCs gene-edited at CCR5, MyD88, or CCR5. i Levels of IL-6 after stimulation with R837 (TLR7) or CpG-A (TLR9) in unprimed and IFN-primed HSPC-pDCs targeted at CCR5, MyD88, or IFNAR1. Data are ±SEM of five (b–d) or three (f–i) donors. Statistical analysis was performed using regular two-way ANOVA followed by Bonferroni post hoc test. Two additional donors are shown in Supplementary Fig. 17