Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of mutations in COL7A1 encoding type VII collagen, the major component of anchoring fibrils which from key attachment structures for dermal-epidermal adhesion. 
To investigate the therapeutic potential of CRISPR/Cas9-mediated gene correction through Homology Directed Repair (HDR) in induced Pluripotent Stem Cells (iPSCs), we have generated iPSCs lines from fibroblasts from 2 RDEB patients homozygous for recurrent COL7A1 mutations in exon 3 or exon 80. 
First, we designed different guide RNAs (gRNAs) targeting the specific mutation or sequences at close distance to the mutation to be corrected (~4 to 20 bp downstream or upstream) for each exon. We selected those with minimal predicted off-target activity. Among the site-specific gRNAs tested, six showed up to 42% cleavage activity in HEK293 cells, as assessed by Sanger sequencing and TIDE analysis. We have also evaluated the off-target activity of these gRNAs in HEK293 cells and found no evidence for non-specific cleavage activity at the in-silico predicted sites. 
To deliver the CRISPR/Cas9 system into RDEB-iPSCs, we tested different nucleofection programs, cell concentrations and compared the efficiency of plasmid to ribonucleoprotein complex (RNP)-based methods. Plasmid delivery of gRNA and Cas9 targeting exon 3 or exon 80 resulted in up to 15% of gene-editing efficiency associated with significant cell toxicity in unselected bulk-nucleofected RDEB-iPSCs. In contrast, RNP-based delivery of gRNA and Cas9 showed less cytotoxicity and up to 32% of targeting efficiency in the same cells, as measured by Sanger sequencing and TIDE analysis.
For gene correction purpose, we treated RDEB-iPSCs with site-specific RNPs together with the corresponding Donor delivered as ssODN.  Up to 20% of genetic correction was observed in bulk-nucleofected RDEB-iPSCs without antibiotic or fluorescent-based selection, as assessed by Sanger sequencing and TIDER analysis. Currently, we are differentiating these cells into keratinocytes and fibroblasts in the perspective of the development of transplantable skin models suitable for clinical application.

CRISPR/Cas9 and Homology Directed Repair-mediated correction of COL7A1 recurrent mutations in induced pluripotent stem cells from patients with recessive dystrophic epidermolysis bullosa