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. 2017 Jan 30;114(7):1660–1665. doi: 10.1073/pnas.1614775114

Fig. S2.

Fig. S2.

In vitro cell culture sgRNA functional test. (A) Schematic diagram of the structure of collagen VII. (B) List of sgRNA molecules tested for Col7a1 exon80 deletion. (C) T7 endonuclease I assay for each sgRNA functional test. (D) PCR amplifications of the Col7a1 genomic DNA demonstrate truncation at exon80 after transfection with different sgRNA pairs. The sgRNA pair used for in vivo Col7a1 gene editing is marked in red. (E) RT-PCR of Col7a1 mRNA indicates deletion at exon80 after transfection with different sgRNA pair combinations. (F) Schematic diagram of the in vitro gene correction assay. Modified pSpCas9-2A-GFP plasmid expresses Cas9 and two different sgRNAs (gRNA-L/R). (G) PCR amplifications of the Col7a1 genomic DNA demonstrate truncation at exon80 after transfection. Percentage of truncated genomic DNA is shown on the Right. (H) Partial Col7a1 gene sequencing result confirms the deletion of exon80. (I) RT-PCR of Col7a1 mRNA indicates deletion of exon80 after transfection. Percentage of truncated mRNA product is shown on the Right. (J) Partial Col7a1 mRNA sequencing result confirms deletion of exon80.