190 Biallelic correction of recessive dystrophic epidermolysis bullosa mutations in iPSCs using CRISPR/Cas9- based genome editing

Abstract

Several approaches for correcting mutations have been reported using sequence-specific nucleases, which allow for efficient genetic modifications at targeted sites of interest. However, the low efficiency drives up the cost and drug selection leads to safety concerns in clinical applications. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7), which is the major constituent of anchoring fibrils at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and thus have severely impaired dermal-epidermal stability. Here, we successfully corrected a recurrent hotspot mutation in exon 19 (c.2470insG) in the COL7A1 gene using homology-directed repair with CRISPR Cas9-gRNA in iPSCs derived from a patient with RDEB who was homozygous for this mutation. We utilized single-strand oligodeoxynuleotides as the donor template, together with a high-fidelity CRISPR/Cas9 nuclease and an mCherry reporter gene to achieve both biallelic and monoallelic correction of COL7A1 mutations, which allowed us to select the targeted cells by FACS without drug selection. The positive mCherry iPSCs were seed in low density in a 60 mm2 cell culture dish to allow iPSC colonies grown from single cells. This strategy resulted in an efficiency of 10% for biallelic correction (WT/WT) and 40% for monoallelic correction (WT/mut). Moreover, Sanger sequence analysis indicated that the target sequence areas were free of undesired mutations. These gene-corrected iPSCs can be differentiated into keratinocytes and fibroblasts for ex vivo 3D skin construction to assess the restoration of C7 protein at the BMZ. Taken together, we have demonstrated correction of specific RDEB mutations without drug selection in a large locus such as COL7A, which is a crucial step for clinical applications to develop innovative stem cell therapies for RDEB.