Targeted chromosomal deletions in human cells using zinc finger nucleases
Targeted chromosomal deletions in human cells using zinc finger nucleases
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DOI：
10.1101/gr.099747.109
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发表时间：
2010-01-01
期刊：
影响因子：
7
通讯作者：
Kim, Jin-Soo
中科院分区：
文献类型：
--
作者：
Lee, Hyung Joo;Kim, Eunji;Kim, Jin-Soo
我们提出了一种利用工程化锌指核酸酶（ZFNs）在人类及其他真核细胞中产生基因组片段靶向缺失的新方法。我们发现，设计用于靶向人类染色体上两个不同位点的ZFNs能够在染色体上引入两个同时发生的DNA双链断裂（DSBs），并导致两个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的频率删除几百个碱基对（bp）到15兆碱基对范围内的预定基因组DNA片段。这些高频率使得我们能够通过有限稀释分离出目标染色体片段已被删除的细胞克隆群。序列分析表明，许多缺失连接点包含小的插入或缺失以及微同源性，这表明是通过非同源末端连接进行的DNA修复。与重组酶和归巢核酸酶等其他基因组工程工具不同，ZFNs不需要将靶点预先插入基因组，并且允许在动物和植物细胞中对内源基因组序列进行精确操作。因此，ZFN诱导的基因组缺失作为一种新方法在生物医学研究、生物技术和基因治疗中应该具有广泛的用途。
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.