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 no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we describe 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 Alzheimer's disease pathogenesis 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 the method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.