Dystrophic Epidermolysis Bullosa (DEB) is a devastating rare disease associated with chronic wounding of the skin. Patients suffer from a wide range of complications including: scarring, blistering, infection, and squamous cell carcinoma. DEB is phenotypically characterized by a lack of functional anchoring fibrils at the dermal-epidermal junction due to mutations in the Collagen type VII Alpha I (COL7A1) gene. Viral gene therapies are being explored for the treatment of DEB. However, viral vectors can elicit an immune response, are susceptible to silencing over time, and can carry a risk of insertional mutagenesis. COL7A1 is a large gene, encompassing 118 exons, several of which are dispensable to protein function. The use of antisense oligonucleotides (ASOs) to cause transient skipping of exon 73 and 80 restored functional anchoring fibrils in patient-derived fibroblasts and keratinocytes. However, ASO-based approaches suffer from limited tissue penetration and require chronic dosing. Gene-editing approaches using TALENs and CRISPR-Cas9 are also being explored for the treatment of DEB. However, current gene-editing approaches suffer from low efficiency, necessitating the use of selection markers that prevent in vivo application. Here we show efficient ablation of the COL7A1 splice acceptor site in primary human keratinocytes and fibroblasts using gene-editing endonucleases delivered via a novel, minimally immunogenic mRNA. We targeted multiple sequences near the exon 73 splice acceptor using TALENs and a novel chromatin- context-sensitive gene-editing endonuclease (NoveSlice). We observed high-efficiency cutting with both endonucleases in a cell-free cleavage assay. We then transfected primary keratinocytes and fibroblasts and characterized editing events using both biased and unbiased methods. The T7 endonuclease I assay confirmed high on-target editing, while RT-PCR of the exon 71-75 region showed durable exclusion of exon 73 from collagen 7 mRNA. Genome-wide off-target screening was conducted to assess potential unintended edit sites. We demonstrate efficient, non-viral ablation of the COL7A1 exon 73 splice acceptor using gene-editing endonucleases delivered via a novel minimally immunogenic mRNA, and we show durable exon exclusion in primary human cells. This approach overcomes the low-efficiency of previous gene-editing approaches, and is being explored for clinical use.