Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.

在这里，我们基于来自4694名韩国人的大规模人群队列数据，通过CNV分析，从3号和8号染色体中找到了两个基因组安全港（GSH）候选者。此外，通过定量实时PCR验证了这些CNVR的估计基因型，并且流行病学数据检查了疾病或性状与两个CNVR之间没有显着的遗传关联。通过计算机方法筛选GSH候选物后，我们设计了TALEN对以将EGFP表达盒整合到人细胞系中，以在体外确认GSH候选物的功能环境。结果，使用TALEN将转基因插入两个基因座之一显示出了与AAVS1位点相当的强大转基因表达，而没有显着干扰邻近基因。改变启动子或细胞类型不会明显干扰这一趋势。因此，我们可以验证两个CNVRs作为在人类细胞中有效和安全地转基因插入的位点。