The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APP^SW allele using clustered regularly interspaced short palindromic repeats (CRISPR). By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APP^SW or APP^WT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APP^SW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APP^SW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof of concept data that CRISPR/Cas9 can selectively disrupt the APP^SW allele both ex vivo and in vivo - and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.

淀粉样蛋白前体蛋白（APP）基因中的APPswe（瑞典语）突变导致淀粉样蛋白-β（Aβ）前体蛋白的β-分泌酶裂解增加，从而导致遗传性阿兹海默氏病（AD）。这不仅在大脑中而且在突变携带者的外周组织中导致异常高的Aβ水平。在这里，我们使用聚类的规则间隔的短回文重复序列（CRISPR）选择性破坏了人类突变体APP ^SW等位基因。通过应用化脓链球菌的CRISPR / Cas9 ，我们产生了APP ^SW或APP ^WT的等位基因特异性缺失。如通过ELISA测量的，靶向的患者来源的成纤维细胞的条件培养基显示分泌的Aβ降低约60％。接下来，将APP ^SW特异性指导RNA（gRNA）和Cas9的编码序列包装到单独的腺相关病毒（AAV）载体中。在从APP ^SW转基因小鼠胚胎（Tg2576）分离的原代神经元中，以及在将这些载体共注射到成年小鼠的海马体中，都实现了位点特异性插入缺失的形成。综上所述，我们在这里提供了概念数据证明，CRISPR / Cas9可以选择性地破坏离体和体内APP ^SW等位基因-从而减少致病性Aβ。因此，该系统可能有可能被开发为针对ADswe和与Aβ升高相关的其他点突变引起的AD基因治疗的工具。