Fig. 1.

Efficient CRIPR/Cas9-mediated integration of IDUA overexpression cassettes into the CCR5 locus in human CD34+ HSPCs. a Schematic of targeted integration of IDUA and expression cassettes. The AAV6 genome was constructed to have 500 bp arms of homology centered on the cut site, and the IDUA sequence placed under the control of the SFFV or the PGK promoter (E = Exon). In two DNA templates, YFP was expressed downstream of IDUA using the self-cleaving P2A peptide. b Representative FACs and histogram plots 3-days post-modification of mock and human HSPCs that underwent RNP and AAV6 exposure with YFP-containing expression cassettes. c Targeting frequencies in cord blood (CB, red dots) and adult peripheral blood (PB, blue dots)-derived HSPCs read by percent fluorescent cells in YFP-expressing cassettes and percent colonies with targeted CCR5 alleles by single cell-derived colony genotyping in cassettes without the reporter. Each dot represents the average of duplicates for a human cell donor. For RNP + AAV6 conditions with YFP templates, n = 20 and n = 11 independent human donors for CB and PB respectively. For the template without selection n = 6 independent human donors in CB and PB. Data shown as mean ± SD. d Distribution of wild type (WT), mono and bi-allelically modified cells (n = 400) in YFP-positive HSPCs