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

Several approaches for correcting mutations have been reported using sequence-specificnucleases, which allow for efficient genetic modifications at targeted sites of interest. How-ever, the low efficiency drives up the cost and drug selection leads to safety concerns inclinical applications. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inheritedskin disorder caused by mutations in theCOL7A1gene 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-epidermalstability. Here, we successfully corrected a recurrent hotspot mutation in exon 19(c.2470insG) in theCOL7A1gene using homology-directed repair with CRISPR Cas9-gRNAin iPSCs derived from a patient with RDEB who was homozygous for this mutation. Weutilized single-strand oligodeoxynuleotides as the donor template, together with a high-fi-delity CRISPR/Cas9 nuclease and an mCherry reporter gene to achieve both biallelic andmonoallelic correction ofCOL7A1mutations, which allowed us to select the targeted cells byFACS without drug selection. The positive mCherry iPSCs were seed in low density in a 60mm2cell culture dish to allow iPSC colonies grown from single cells. This strategy resulted inan 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 werefree of undesired mutations. These gene-corrected iPSCs can be differentiated into kerati-nocytes and fibroblasts forex vivo3D skin construction to assess the restoration of C7 proteinat the BMZ. Taken together, we have demonstrated correction of specific RDEB mutationswithout drug selection in a large locus such asCOL7A,which is a crucial step for clinicalapplications to develop innovative stem cell therapies for RDEB.