Figure 1.

COL7A1 E80 Dual RNA Guide CRISPR/Cas9 Deletion Strategy

(A) Scheme of the strategy. Single CRISPR RNA guides were designed to induce Cas9-mediated DNA double-strand breaks within selected COL7A1 intron sequences flanking exon 80 (U-Guides and D-Guides). NHEJ repair leads to intron-intron rejoining with concomitant E80 deletion and restoration of COL7A1 reading frame and truncated C7 expression. (B) CRISPR guide design. sgRNA (sgRNAs 1, 2, 3, and 4) sequences and alignment to E80-flanking sequences E80 are shown. The protospacer adjacent motif (PAM) is indicated in a darker color. (C) Predicted amplicon sizes for E80 deletions corresponding to each sgRNA pair combination. (D) PCR analysis of genomic DNA from RDEB keratinocytes from patient P1 treated with the RNP complexes containing the different sgRNA pair combinations. The sg2 + sg3 pair yielded the highest proportion of excised E80 according to the intensity of the lower 440-bp band. Deletion ratios assessed by densitometry are shown in each lane. “A” and “B” refer to the two different electroporation conditions tested.