Elimination of the extra chromosome of Dup15q syndrome iPSCs for cellular and molecular investigation.

ABSTRACT

Chromosome 15q11.2-13.1 duplication (Dup15q) syndrome is one of the most common autism spectrum disorders (ASDs) associated with copy number variants (CNVs). For the analysis of CNV-relevant pathological cellular phenotypes, a CNV-corrected isogenic cell line is useful for excluding the influence of genetic background. Here, we devised a strategy to remove the isodicentric chromosome 15 by inserting a puro-ΔTK selection cassette into the extra chromosome using the CRISPR-Cas9 system, followed by a subsequent two-step drug selection. A series of assays, including qPCR-based copy number analysis and karyotype analysis, confirmed the elimination of the extra chromosome. Furthermore, cerebral organoids were generated from the parental Dup15q iPSCs and their isogenic iPSCs. scRNA-seq analysis revealed the alteration of expression levels in ion-channel-related genes and synapse-related genes in glutamatergic and GABAergic neurons in Dup15q organoids, respectively. The established isogenic cell line is a valuable resource for unraveling cellular and molecular alterations associated with Dup15q syndrome.