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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases
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Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases

机译：使用设计的锌指核酸酶将基因靶向添加到人类基因组中的指定位置

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摘要

Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.
机译：尽管将其有效地应用于遗传医学，生物技术和基础研究，将新的DNA序列有效地整合到人类细胞基因组中的特定位点仍然是一个挑战。我们发现，由工程锌指核酸酶（ZFNs）诱导的精确放置的双链断裂可以刺激长DNA片段的整合到预定的基因组位置，从而导致高效的位点特异性基因添加。使用带有一个12 bp标签，一个900 bp ORF或一个1.5 kb启动子转录单位并位于基因座特异性同源臂两侧的染色体外DNA供体，我们发现目标整合频率为15％，6％和5％分别在治疗72小时之内，并且没有针对所需事件进行选择。重要的是，我们发现整合事件以同源性指导的方式发生，并导致内源染色体基因座上供体指定基因型的准确重建，因此推测是由于使用供体的合成依赖性链退火修复断裂而导致的以DNA为模板。发生这种位点特异性基因，随机整合率没有可测量的增加。值得注意的是，我们还发现ZFNs可以在没有任何选择的情况下，以6％的频率将带有三个不同启动子转录单元的8kb序列添加到内源性基因座中。这些数据揭示了参与双链断裂修复的特殊聚合酶机制令人惊讶的多功能性，阐明了哺乳动物细胞工程的有效方法，并为人类遗传性疾病打开了ZFN驱动的基因添加疗法的可能性。

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