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

Abstract

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.

• Authors: Tao Ye, Yangyang Duan, Hayley W S Tsang, He Xu, Yuewen Chen, Han Cao, Yu Chen, Amy K Y Fu, Nancy Y Ip
• Journal: Communications biology (Commun Biol) Vol. 4 Issue 1 Pg. 195 (02 12 2021) ISSN: 2399-3642 [Electronic] England
• PMID: 33580208 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• APP protein, human
• Amyloid beta-Peptides
• Amyloid beta-Protein Precursor
• MAPT protein, human
• tau Proteins
• CRISPR-Associated Protein 9

Topics

• Amyloid beta-Peptides (metabolism)
• Amyloid beta-Protein Precursor (genetics, metabolism)
• Apoptosis
• CRISPR-Associated Protein 9 (genetics, metabolism)
• CRISPR-Cas Systems
• Cell Line
• Clustered Regularly Interspaced Short Palindromic Repeats
• DNA Copy Number Variations
• Gene Dosage
• Gene Editing
• Gene Expression Regulation
• Humans
• Induced Pluripotent Stem Cells (metabolism, pathology)
• Neurogenesis
• Neurons (metabolism, pathology)
• Phosphorylation
• Proof of Concept Study
• tau Proteins (metabolism)