Program in Neuroscience, Department of Biomedical Sciences, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL, 32306, USA.
Sci Rep. 2023 May 19;13(1):8109. doi: 10.1038/s41598-023-35203-7.
The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.
突变小鼠的创建对于推进生物医学科学是非常宝贵的，但对于研究全范围的突变和多态性来说，时间和资源过于密集。因此，细胞培养模型是小鼠模型的宝贵补充，特别是对于生物钟等细胞自主途径。在这项研究中，我们定量评估了使用 CRISPR 在小鼠胚胎成纤维细胞 (MEF) 中创建细胞模型与在小鼠模型中的应用。我们使用相同的 sgRNA 和 HDR 修复模板在小鼠和 MEF 中生成时钟基因 Per1 和 Per2 的两个点突变，并通过数字 PCR 定量突变的频率。与 MEF 相比，在小鼠受精卵中的突变频率高了一个数量级。然而，MEF 中的突变频率仍然足够高，可以通过简单筛选几十个单个细胞进行克隆分离。我们生成的 Per 突变细胞为 PAS 结构域在调节 PER 磷酸化中的作用提供了重要的新见解，这是生物钟机制的一个关键方面。批量 MEF 群体中突变频率的定量为优化 CRISPR 方案和时间/资源规划提供了有价值的基础，以便进一步研究生成细胞模型。
