Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.

隐性营养不良性表皮松解性大疱是一种罕见且严重的遗传性皮肤病，可导致皮肤和粘膜起泡。隐性营养不良性表皮松解症（RDEB）由各种各样的突变引起COL7A1 -encoding VII型胶原蛋白，其是用于真皮-表皮粘附必不可少的。在这里，我们证明了离体COL7A1的可行性通过CRISPR / Cas9介导的同源性指导的修复在原代RDEB细胞和移植的3D皮肤等效物中进行编辑。我们设计了五个指导RNA来纠正外显子2中的RDEB致病性无效突变（c.189delG; p.Leu64Trpfs * 40）。在测试的位点特异性指导RNA中，当以整合缺陷型慢病毒形式交付时，一种在原代RDEB角质形成细胞和成纤维细胞中显示出显着的裂解活性。通过等位基因特异性高度敏感的TaqMan液滴数字PCR（ddPCR）在转导的角质形成细胞和成纤维细胞进行了遗传校正，从而导致11％和修正的15.7％COL7A1mRNA表达分别，无需抗生素选择。经过基因校正的3D皮肤等效物嫁接到裸鼠上，显示出高达26％的重新表达和VII型胶原蛋白的正常定位，以及在真皮-表皮交界处锚定原纤维形成。我们的研究提供了证据，即在原代RDEB细胞中进行精确的基因组编辑是从遗传角度校正COL7A1突变以开发未来离体临床应用的一种相关策略。