Figure 2. c.553 COL7A1 base editing in RDEB primary cells.

a and b Quantification of DNA base editing by deep sequencing. Genomic DNA and mRNA were sequenced on an Illumina MiSeq to determine the frequency of A:T>G:C editing of a c.553 cells and b c.1573 cells. Values and error bars are the mean and standard deviation from five experimental replicates. sgRNA sequences for each mutation are shown and the red lettered ‘TGA’ represents the nonsense mutation codon. Superscript numbers represent the target base for mutation correction or bystander editing and are numbered relative to the 5’ start of the sgRNA. c Immunofluorescence of primary fibroblasts. White boxes embedded in the images on the left identify samples in that row. Labels at top identify the antigen stained for in that column. Edited and uncorrected cells were stained simultaneously with equivalent amounts of anti-vimentin and anti-collagen type VII polyclonal antibodies. The images for each fluorescent channel were merged with DAPI nuclear stain that is shown at right. Images are representative of three independent experiments. Scale bar=50 μm (lower right in WT vimentin image). d and e C7 Western blotting. d Cell lysates from uncorrected 553 and 1573 cells were analyzed in parallel with base edited 553 and 1573 cells using a polyclonal anti-C7 antibody. Wild type (WT) cells are from a healthy donor. The C7 lane shows the ~290 kD C7 band and actin was used as a loading control. e Secreted C7 from cell supernatant. C7 was detected in the supernatant of c.553 edited cells that were plated in serum free media. WT is a healthy donor lysate sample and negative control (ctrl) is from cells that have a COL7A1 mutation that inactivates the gene. Pro-collagen type VII is shown at 290 kD with yellow arrows. A larger molecular weight species is shown with a white arrow representing the non-reduced C7 ultrastructural trimeric polypeptide. The Ponceau S loading control is shown and is labeled ‘PS.’