Finlay Darren, Hor Pooja, Goldenson Benjamin H, Li Xiao-Hua, Murad Rabi, Kaufman Dan S, Vuori Kristiina
NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037, USA.
Sanford Stem Cell Institute, University of California-San Diego, La Jolla, California 92037, USA.
bioRxiv. 2025 May 11:2025.05.10.653237. doi: 10.1101/2025.05.10.653237.
We demonstrate here the use of optical genome mapping (OGM) to detect genetic alterations arising from gene editing by various technologies in human induced pluripotent stem cells (iPSCs). OGM enables an unbiased and comprehensive analysis of the entire genome, allowing the detection of genomic structural variants (SVs) of all classes with a quantitative variant allele frequency (VAF) sensitivity of 5%. In this pilot study, we conducted a comparative dual analysis between the parental iPSCs and the derived cells that had undergone gene editing using various techniques, including transposons, lentiviral transduction, and CRISPR-Cas9-mediated safe harbor locus insertion at the adeno-associated virus integration site 1 (AAVS1). These analyses demonstrated that iPSCs that had been edited using transposons or lentiviral transduction resulted in a high number of transgene insertions in the genome. In contrast, CRISPR-Cas9 technology resulted in a more precise and limited transgene insertion, with only a single target sequence observed at the intended locus. These studies demonstrate the value of OGM to detect genetic alterations in engineered cell products and suggests that OGM, together with DNA sequencing, could be a valuable tool when evaluating genetically modified iPSCs for research and therapeutic purposes.
我们在此展示了使用光学基因组图谱（OGM）来检测人类诱导多能干细胞（iPSC）中各种技术进行基因编辑所产生的基因改变。OGM能够对整个基因组进行无偏且全面的分析，可检测所有类型的基因组结构变异（SV），其定量变异等位基因频率（VAF）灵敏度为5%。在这项初步研究中，我们对亲代iPSC与使用各种技术进行基因编辑后的衍生细胞进行了比较性双重分析，这些技术包括转座子、慢病毒转导以及CRISPR - Cas9介导的在腺相关病毒整合位点1（AAVS1）进行安全港位点插入。这些分析表明，使用转座子或慢病毒转导编辑的iPSC在基因组中导致了大量转基因插入。相比之下，CRISPR - Cas9技术导致了更精确且有限的转基因插入，在预期位点仅观察到单个靶序列。这些研究证明了OGM在检测工程细胞产品基因改变方面的价值，并表明OGM与DNA测序一起，在评估用于研究和治疗目的的基因编辑iPSC时可能是一种有价值的工具。
