Takahashi Gou, Maeda Minato, Shinozaki Kayoko, Harada Gakuro, Ito Saburo, Miyaoka Yuichiro
Regenerative Medicine Project, Tokyo Metropolitan Institute of Medical Science, Setagaya, Tokyo, 156-8506, Japan.
Department of Animal Science, Tokyo University of Agriculture, Atsugi, 243-0034, Kanagawa, Japan.
Stem Cell Res Ther. 2025 Jun 7;16(1):295. doi: 10.1186/s13287-025-04414-2.
Genome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool, but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore, genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations, but not in single cells. However, systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells, it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.
In this study, we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps, which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.
We analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations, including insertions and deletions, such as 1-bp insertions and 8-bp deletions. Moreover, we successfully cryopreserved and then recovered genome-edited iPS cell clones, demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.
This study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.
人类诱导多能干细胞（iPS细胞）中的基因组编辑是再生医学中的一种强大方法。CRISPR-Cas9是最常用的基因组编辑工具，但除了所需的编辑外，它还经常诱导副产物插入和缺失。因此，iPS细胞的基因组编辑会产生多种基因型。现有的检测方法大多分析细胞群体中的基因组编辑结果，而非单个细胞中的结果。然而，缺乏对单个iPS细胞中基因组编辑结果的系统分析。由于人类iPS细胞作为分离的单个细胞时死亡率很高，因此很难高通量分析基因组编辑的iPS细胞克隆。
在本研究中，我们开发了一种基于精确机器人挑选源自细胞外基质中单个细胞生长形成的细胞团块的高通量iPS细胞克隆分离方法。我们首先通过CRISPR-Cas9将点突变引入人类iPS细胞库。这些经过基因组编辑的人类iPS细胞被解离并作为单个细胞在细胞外基质中培养以形成细胞团块，然后使用细胞操作机器人将其分离以建立经过基因组编辑的人类iPS细胞克隆。通过扩增子测序分析这些克隆中的基因组编辑结果，以确定各个iPS细胞克隆的基因型。在确定其基因型的同时，我们识别并区分了由CRISPR-Cas9诱导的不同插入和缺失的序列。我们还冷冻保存了已建立的iPS细胞克隆，并在确定其基因型后将其复苏。
我们分析了1000多个经过基因组编辑的iPS细胞克隆，发现纯合编辑比杂合编辑更为频繁。我们还观察到相同基因操作（包括插入和缺失，如1个碱基对的插入和8个碱基对的缺失）的纯合诱导现象很常见。此外，我们成功冷冻保存并随后复苏了经过基因组编辑的iPS细胞克隆，表明我们基于细胞操作机器人的方法在建立经过基因组编辑的iPS细胞克隆方面具有价值。
本研究揭示了人类iPS细胞基因组编辑中一个先前未知的特性，即单个细胞中目标序列的两个拷贝倾向于诱导相同的序列操作。我们的新克隆方法和研究结果将促进基因组编辑在人类iPS细胞中的应用。
