The dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause of autosomal dominant diseases such as Alzheimer’s disease. However, there is currently no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we demonstrate a simple and precise method to simultaneously generate iPSC lines with different gene dosages using paired Cas9 nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motif specificity to expand the targetability of double-nicking–mediated genome editing. As a proof-of-concept study, we examine the gene dosage effects on an Alzheimer’s disease patient-derived iPSC line that carries three copies of APP (amyloid precursor protein). This method enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically corrected iPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes of amyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCs with different gene dosages using our method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.

由于重复或单倍体不足导致的基因剂量失调是常染色体显性遗传疾病（如阿尔茨海默病）的主要原因。然而，目前还没有在人类模型系统如人类诱导多能干细胞 (iPSCs) 中操纵基因剂量的快速有效方法。在这里，我们展示了一种使用成对 Cas9 切口酶同时生成具有不同基因剂量的 iPSC 系的简单而精确的方法。我们首先生成具有更广泛的原型间隔区相邻基序特异性的 Cas9 切口酶变体，以扩展双切口介导的基因组编辑的靶向性。作为概念验证研究，我们检查了基因剂量对阿尔茨海默病患者来源的 iPSC 系的影响，该 iPSC 系携带三个拷贝的APP（淀粉样前体蛋白）。这种方法可以快速和同时生成具有单等位基因、双等位基因或三等位基因敲除APP的 iPSC 系。由等基因校正的 iPSC 产生的皮层神经元表现出对淀粉样蛋白 β 分泌和 Tau 过度磷酸化的疾病相关表型的基因剂量依赖性校正。因此，使用本文所述的方法快速生成具有不同基因剂量的 iPSC 可以成为研究疾病机制和治疗发展的有用模型系统。