Xing Jiani, Zhang Cunfang, Xu Kun, Hu Linyong, Wang Ling, Zhang Tingting, Ren Chonghua, Zhang Zhiying
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, USA.
Methods Mol Biol. 2018;1867:175-183. doi: 10.1007/978-1-4939-8799-3_13.
Using engineered nucleases such as zinc-finger nucleases (ZFNs) and TALE nuclease (TALEN) to accomplish genome editing often causes high cellular toxicity because of the consistent expression of artificial nucleases and off-targeting effect. And lacking selection marker in modified cells makes it hard to enrich these positive cells. Here we introduce a method by incorporating a surrogate reporter enrichment into a suicidal ZFN system, which is designed by a pair of ZFN expression cassettes flanked with its target sites. Our data demonstrated that this modified system achieved almost the same ZFN activity as the original method but reduced ~40% toxicity. This new suicidal ZFN expression system coupled with a surrogate reporter not only enables decreased cellular toxicity but also makes the genetic modified cells to be enriched by EGFP analysis.
使用诸如锌指核酸酶（ZFNs）和转录激活因子样效应物核酸酶（TALEN）等工程核酸酶来完成基因组编辑，常常会因为人工核酸酶的持续表达和脱靶效应而导致较高的细胞毒性。而且修饰细胞中缺乏选择标记使得富集这些阳性细胞变得困难。在此，我们介绍一种方法，即将替代报告基因富集整合到自杀性ZFN系统中，该系统由一对侧翼带有其靶位点的ZFN表达盒设计而成。我们的数据表明，这种修饰后的系统实现了与原始方法几乎相同的ZFN活性，但毒性降低了约40%。这种新的自杀性ZFN表达系统与替代报告基因相结合，不仅能够降低细胞毒性，还能通过绿色荧光蛋白（EGFP）分析富集基因修饰细胞。
