Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases

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.