Figure 2.

AAV transduction and genotyping of RDEB keratinocytes after AAV and RNP treatment

(A) Transduction efficacy of eight different AAV serotypes in primary keratinocytes. Percentage of GFP⁺ cells determined by flow cytometry for each serotype is shown. AAV6 donor vector was used at a an MOI of 30,000. Mean ± SD (bars) are shown. (B) Common mutations in COL7A1 region contained in AAV donor template constructs; exons are represented as red boxes. Symmetrical (Sym) and asymmetrical (Asym) vector designs are shown; exons are represented as blue boxes. Primers used for PCR genotyping were F (out) and R (in) (purple arrows). (C) PCR genotyping in primary RDEB keratinocytes treated with the gene-editing protocol. Duplicate experiments for each condition were performed. Upper (filled arrowhead) and lower (open arrowhead) bands correspond to non-recombinant and intron 79-lacking recombinant alleles, respectively; recombinants were found only in cells treated with both AAV-donor template and CRISPR-Cas9 RNP. Frequencies of HDR are shown below. R1, R2, experimental replicates 1 and 2.