Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
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DOI：
10.1038/s41598-018-36736-y
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发表时间：
2019-01-22
影响因子：
4.6
通讯作者：
Lee, Choogon
中科院分区：
文献类型：
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作者：
Jin, Yuan-Hu;Joo, Hyunjeong;Lee, Choogon
CRISPR-Cas9是一种强大的基因编辑技术，可以在几乎任何哺乳动物细胞系中诱导目标基因突变。然而，如果靶细胞系难以转染并且不增殖，则其实用性可能受到限制。在目前的研究中，我们开发了一种基于CRISPR的基因敲除的简化方法，具有三个关键优势，它允许在没有克隆选择和扩增的情况下进行基因敲除的表型测定。首先，它整合到Cas9、sgRNA和荧光标记物的单一多合一载体转基因中。其次，我们使用Gateway系统通过PCR和体外重组将特异性sgRNA快速克隆到一体化载体中，而无需常规的酶消化和连接。第三，它使用腺病毒包装一体化载体的能力，以及其高转导效率。我们在生物钟模型细胞系U2 OS中测试了一体化腺病毒CRISPR-Cas9，并证明了必需的时钟基因如Bmal 1和Per 1被如此有效地敲除，以至于可以在没有任何克隆选择和扩增的情况下从异源群体中进行功能测定。这种简化的方法可能被证明是非常宝贵的候选基因在不同的生物途径，包括昼夜节律钟的快速功能测定。
CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.