Figure 5.

CRISPR/Cas9-Induced Therapeutic Rearrangements in HSPCs by RNP-Mediated Delivery

(A) IF representative picture of Cas9⁺ cord blood-derived HSPCs 6 hr after RNP electroporation. Original magnification, 40×. Scale bar, 200 μm. (B) Cleavage efficiency of individual gRNAs in adult HSPCs. Error bars denote SD. (C) Frequency of genomic modifications in erythroblasts derived from adult HSPCs electroporated using Cas9 RNP complexes containing gRNA pairs. Error bars denote SD. (D) Frequency of CFC in RNP-electroporated adult HSPCs. Untreated (UT), mock-transfected (Mock), and Cas9-only-transfected HSPCs served as controls (n = 4–8 donors with at least two replicates/donor). **p < 0.01, ***p < 0.001 (two-way ANOVA plus Tukey’s multiple comparison test). Error bars denote SD. (E) Correlation between frequency of genomic rearrangements and fold change in the proportion of F cells in edited cells compared to control samples. R², line-of-best-fit equation and p value are indicated. For the Corfu-edited samples, we removed an outlier from the trend line (indicated with a gray circle).