End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

基于末端连接的基因编辑通常用于有效重组和敲除靶基因。但是，相关的随机，不可预测且通常是异类的修复结果限制了其在治疗方法中的适用性。这项研究仅基于CRISPR / Cas9靶位点的序列背景揭示了更精确和可预测的结果。疱疹性皮肤病大疱性表皮松解症（DEB）的严重营养不良形式代表了一个合适的模型平台，可以分别测试这些最近的发展，以分别对优势和隐性等位基因进行破坏和重组，这两种情况都经常在DEB中看到。我们将CRISPR / Cas9核酸酶作为核糖核蛋白递送到了原代野生型和隐性DEB角质形成细胞中，以在目标部位引入精确可预测的单腺嘌呤有义链插入。我们在40％以上的核糖核蛋白治疗的原始野生型角质形成细胞中实现了VII型胶原基因敲除，在70％以上的核糖核蛋白治疗的隐性DEB角质形成细胞中实现了VII型胶原蛋白的修复。靶点的下一代测序表明，至少有17％的被分析者在病原体突变的上游存在精确的腺嘌呤插入COL7A1等位基因。这表明基于精确的末端连接介导的DNA修复的COL7A1编辑是一种有效的策略，无论突变遗传如何，均可逆转DEB的疾病相关性。