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. 2019 Mar 15;27(5):986–998. doi: 10.1016/j.ymthe.2019.03.007

Figure 5.

Figure 5

Collagen VII Expression in ΔE80 RDEB Polyclonal Keratinocytes

Keratinocytes were treated with the RNP complex containing different CRISPR dual sgRNA combinations, and C7 expression was assessed by immunofluorescence (IF) and western blot. (A) C7 IF analysis. Left top: control, untreated RDEB (c.6527insC) keratinocytes are shown. Right top: RDEB keratinocytes were treated with the sg2 + sg4 pair. Left bottom: RDEB keratinocytes were treated with the sg1 + sg3 pair. Right bottom: RDEB keratinocytes were treated with the sg2 + sg3 pair. DAPI was used to stain nuclei. Scale bars, 50 μm. (B) Western blot analysis of C7 expression in unedited and edited RDEB keratinocytes, showing C7 bands intensities consistent with the IF data. (C) Western blot analysis of secreted C7 from culture supernatant of normal, untreated RDEB and the sg2 + sg3 RNP-treated RDEB keratinocytes. Protein loading was assessed by Ponceau red staining of the membrane (bottom). The ΔE80 C7 in edited RDEB cells is indistinguishable from that of normal human keratinocytes.