FIGURE 3.

HDT conjugation enhances PU-PEI₆₀₀-Mal-mediated co-delivery of CRISPR/Cas9 components. (A) Fluorescence microscopy images of CM-iPSCs treated with the unconjugated mix of PU-PEI₆₀₀-Mal/plasmid1 and PU-PEI₆₀₀-Mal/plasmid2 complexes (top) and HDT-conjugated complexes (bottom) showing the expression of GFP (green) and mCherry (red). (B) Schematic outline of GLA gene and mRNA products of its wild type and IVS4 + 919 G > A mutant forms with the indicated positions of forward (F) and reverse (R) primers designed to specifically differentiate between them. (C) RT-PCR analysis of the expression of wild type (217 bp amplicon) and IVS4 + 919 G > A (272 bp amplicon) GLA forms in CM-iPSCs treated with the indicated PU-PEI₆₀₀ CRISPR/Cas9 carrier complexes. (D) Quantification of band intensity of RT-PCR electrophoregrams (C) and calculated percentage of mutant to wild type correction. Three independent experiments were performed, and mean values are shown with standard deviation error bars. ^∗∗p < 0.001 (Student’s t-test). (E) Sanger sequencing showing A→G correction of IVS4 + 919 G > A genotype in FC-iPSCs treated with PU-PEI₆₀₀-Mal-HDT carrying CRISPR/Cas9 components (bottom). The sequencing of the original FC-iPSC clone shown on top.