Quantification of Zinc Finger Nuclease-Associated Toxicity
Quantification of Zinc Finger Nuclease-Associated Toxicity
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DOI：
10.1007/978-1-60761-753-2_14
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发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Cathomen, Toni
中科院分区：
文献类型：
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作者：
Cornu, Tatjana I.;Cathomen, Toni
用于靶向基因组编辑的人工锌指核酸酶（ZFN）的最新发展为生物技术和基因治疗开辟了广泛的可能性。ZFN技术允许研究人员在复杂的基因组中故意选择靶位点，并创建适当的核酸酶以在该位点插入DNA双链断裂（DSB）。在非选择的人类细胞中，基因编辑频率高达50%，这证明了这项技术的强大。应用ZFN的潜在副作用包括由于在脱靶位点处的裂解而引起的毒性。这可能是由于DNA结合的特异性不足，因此允许ZFN在基因组内的相似靶序列处具有活性，或者在核酸酶与DNA正确结合之前激活ZFN核酸酶结构域。在这里，我们描述了两种不同的方法来量化ZFN相关的毒性：遗传毒性试验是基于ZFN诱导的DSB修复灶的量化，而细胞毒性是基于应用ZFN后评估细胞存活率。
The recent development of artificial zinc finger nucleases (ZFNs) for targeted genome editing has opened a broad range of possibilities in biotechnology and gene therapy. The ZFN technology allows a researcher to deliberately choose a target site in a complex genome and create appropriate nucleases to insert a DNA double-strand break (DSB) at that site. Gene editing frequencies of up to 50% in non-selected human cells attest to the power of this technology. Potential side effects of applying ZFNs include toxicity due to cleavage at off-target sites. This can be brought about by insufficient specificity of DNA binding, hence allowing ZFN activity at similar target sequences within the genome, or by activation of the ZFN nuclease domains before the nuclease is properly bound to the DNA. Here, we describe two different methods to quantify ZFN-associated toxicity: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based on assessing cell survival after application of ZFNs.