Department of Chemistry, Seoul National University, Seoul, South Korea.
Genome Res. 2010 Jan;20(1):81-9. doi: 10.1101/gr.099747.109. Epub 2009 Dec 1.
We present a novel approach for generating targeted deletions of genomic segments in human and other eukaryotic cells using engineered zinc finger nucleases (ZFNs). We found that ZFNs designed to target two different sites in a human chromosome could introduce two concurrent DNA double-strand breaks (DSBs) in the chromosome and give rise to targeted deletions of the genomic segment between the two sites. Using this method in human cells, we were able to delete predetermined genomic DNA segments in the range of several-hundred base pairs (bp) to 15 mega-bp at frequencies of 10(-3) to 10(-1). These high frequencies allowed us to isolate clonal populations of cells, in which the target chromosomal segments were deleted, by limiting dilution. Sequence analysis revealed that many of the deletion junctions contained small insertions or deletions and microhomologies, indicative of DNA repair via nonhomologous end-joining. Unlike other genome engineering tools such as recombinases and meganucleases, ZFNs do not require preinsertion of target sites into the genome and allow precise manipulation of endogenous genomic scripts in animal and plant cells. Thus, ZFN-induced genomic deletions should be broadly useful as a novel method in biomedical research, biotechnology, and gene therapy.
我们提出了一种新的方法，使用工程化的锌指核酸酶（ZFN）在人类和其他真核细胞中生成靶向基因组片段的缺失。我们发现，设计用于靶向人类染色体上两个不同位点的 ZFN 可以在染色体中引入两个同时发生的 DNA 双链断裂（DSB），并导致两个靶位点之间的基因组片段发生靶向缺失。在人类细胞中使用这种方法，我们能够以 10(-3) 到 10(-1)的频率删除几个至数百个碱基对（bp）到 15 兆碱基对（Mb）范围内的预定基因组 DNA 片段。这些高频率使我们能够通过限制稀释分离出靶染色体片段缺失的克隆细胞群体。序列分析表明，许多缺失连接点含有小的插入或缺失和微同源性，表明通过非同源末端连接进行 DNA 修复。与其他基因组工程工具（如重组酶和 meganuclease）不同，ZFN 不需要将靶位点预先插入基因组中，并允许在动植物细胞中精确操作内源性基因组序列。因此，ZFN 诱导的基因组缺失应该作为一种新的方法在生物医学研究、生物技术和基因治疗中广泛应用。
