Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes.
Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes.
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DOI：
10.1038/s41598-023-35203-7
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发表时间：
2023-05-19
影响因子：
4.6
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中科院分区：
文献类型：
--
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突变小鼠的创造对于推进生物医学科学是非常宝贵的，但对于研究全方位的突变和多态性来说，时间和资源过于密集。因此，细胞培养模型是对小鼠模型的宝贵补充，特别是对于细胞自主途径，如生物钟。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）中创建细胞模型与小鼠模型的比较。我们使用相同的sgRNA和HDR修复模板在小鼠和MEFs中的时钟基因Per1和Per2中产生了两个点突变，并通过数字PCR定量了突变的频率。小鼠受精卵中的频率比MEFs中的频率高约一个数量级。然而，MEFs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。我们产生的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。大量MEF群体中突变频率的定量为优化CRISPR方案和时间/资源规划以生成用于进一步研究的细胞模型提供了有价值的基础。
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.
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影响因子：
9.8
作者：
Hennig S;Strauss HM;Vanselow K;Yildiz O;Schulze S;Arens J;Kramer A;Wolf E
通讯作者：
Wolf E
影响因子：
4.6
作者：
Maggio I;Holkers M;Liu J;Janssen JM;Chen X;Gonçalves MA
通讯作者：
Gonçalves MA
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
4.6
作者：
Kim B;Kim J;Chun M;Park I;Kwak D;Choi M;Kim K;Choe HK
通讯作者：
Choe HK
影响因子：
4.6
作者：
Jin, Yuan-Hu;Joo, Hyunjeong;Lee, Choogon
通讯作者：
Lee, Choogon