The prokaryotic CRISPR/Cas9 system has recently emerged as a powerful tool for genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this genome editing machinery and indeed the very feasibility of using these techniques in vivo remain challenging for most tissue types. Here, we show that nonreplicable Cas9/sgRNA ribonucleoproteins can be used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. We developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatal mice. This method results in rapid gene editing in epidermal stem cells. Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mouse model, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment. This study provides an in vivo gene correction strategy using ribonucleoproteins as curative treatment for genetic diseases in skin and potentially in other somatic tissues.in vivo gene editing | Cas9/sgRNA ribonucleoproteins | skin stem cell | electroporation | RDEB C RISPR/Cas9-mediated genome editing has recently emerged as a powerful tool for genome engineering and for use as a potential therapeutic method to treat patients with genetic diseases (1-3). CRISPR/Cas9-mediated somatic genome editing of multiple mice organs, including lung, liver, brain, pancreas, and muscle, have been reported (4-10). To date, most approaches have relied on virus-based delivery systems, which have drawbacks such as potential integration of viral DNA into a host genome, off-target effects due to prolonged expression of genome editing machinery, and possible activation of virus-triggered host immune responses. Additionally, most therapeutic applications will require the tissue-specific delivery of the genome editing machinery in vivo. So far, this has remained challenging for most tissues, such as skin.In healthy skin, epidermal keratinocytes and dermal fibroblasts secrete collagen VII protein and it in turn forms stable homotrimers to assemble into networks of anchoring fibrils (11,12). The anchoring fibrils are located within the basement membrane zone (BMZ) between the epidermis and dermis, where they participate in stabilizing the association of the epidermis to the underlying dermis (13). In patients with recessive dystrophic epidermolysis bullosa (RDEB), mutations in the Col7a1 gene cause absent or dysfunctional collagen VII protein production, which leads to defective epidermal-dermal adhesion (14). The main clinical manifestations of RDEB include: chronic and severe cutaneous blistering, especially on hands and feet; damage to internal epithelia, such as oral, esophageal, and anal structures; an increased risk for developing aggressive forms of squamous cell carcinoma; and overall reduced life expectanc...
