Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; Takeda-CiRA (T-CiRA) Joint Program, 2-26-1, Muraoka-Higashi, Fujisawa 251-8555, Japan.
Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; Takeda-CiRA (T-CiRA) Joint Program, 2-26-1, Muraoka-Higashi, Fujisawa 251-8555, Japan; iPSC-based Drug discovery and Development Team, RIKEN BioResource Research Center, 1-7 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan; Medical-Risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Eur J Cell Biol. 2024 Sep;103(3):151446. doi: 10.1016/j.ejcb.2024.151446. Epub 2024 Jul 18.
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.
15q11.2-13.1 号染色体重复（Dup15q）综合征是最常见的与拷贝数变异（CNVs）相关的自闭症谱系障碍（ASD）之一。为了分析与 CNV 相关的病理性细胞表型，CNV 校正的同基因细胞系对于排除遗传背景的影响很有用。在这里，我们设计了一种策略，使用 CRISPR-Cas9 系统将 puromycin-ΔTK 选择盒插入多余的染色体，从而去除等臂染色体 15，随后进行两步药物选择。一系列检测，包括基于 qPCR 的拷贝数分析和核型分析，证实了多余染色体的消除。此外，还从 Dup15q iPSC 及其同基因 iPSC 中生成了大脑类器官。scRNA-seq 分析分别揭示了 Dup15q 类器官中谷氨酸能和 GABA 能神经元中离子通道相关基因和突触相关基因表达水平的改变。所建立的同基因细胞系是揭示 Dup15q 综合征相关细胞和分子改变的有价值资源。
