Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system
Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system
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DOI：
10.1007/s10616-018-0252-2
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发表时间：
2018-09
期刊：
影响因子：
2.2
通讯作者：
Ya Li;Weifeng Zhang;Junli Zhao;Sai Li;Linlin Shan;Jiuling Zhu;Yan Li;He Zhu;Q. Mao;
中科院分区：
文献类型：
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作者：
Ya Li;Weifeng Zhang;Junli Zhao;Sai Li;Linlin Shan;Jiuling Zhu;Yan Li;He Zhu;Q. Mao;
成簇调节性散布短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统已被广泛用于基因敲除。慢病毒载体通常用作该系统的递送方法，然而，由于慢病毒整合而延长的Cas9/sgRNA表达可导致累积的脱靶突变。为了解决基因敲除细胞系工程中的这一问题，本研究建立了一个由两个慢病毒载体组成的新系统。一种慢病毒载体同时携带靶向单个或多个基因的sgRNA和CRISPR/Cas9表达盒;另一种慢病毒载体携带Cre，其可以在实现基因靶向后去除基因组中过量的sgRNA和Cas9表达盒。为了证明这一原理，选择了两个候选基因，细胞外基质蛋白1（ECM 1）和颗粒蛋白前体（PGRN），这两个基因都在MDA-MB-231细胞中高度表达，用于测试新系统。在MDA-MB-231细胞系中成功实现了ECM 1和PGRN的双敲除，其中通过Cre去除了sgRNA和Cas9表达盒。该系统在体外多基因敲除方面具有很大的应用潜力。
The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.