Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.
Determining the specificities of TALENs, Cas9, and other genome-editing enzymes.
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DOI：
10.1016/b978-0-12-801185-0.00003-9
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发表时间：
2014
影响因子：
--
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Pattanayak, Vikram;Guilinger, John P.;Liu, David R.
可编程位点特异性核酸内切酶的快速发展导致了用于研究和治疗目的的基因组工程活动的急剧增加。现在可以使用锌指核酸酶（ZFN）、转录激活因子样内切核酸酶（TALEN）和CRISPR相关的Cas9内切核酸酶以位点特异性方式修饰包括哺乳动物在内的广泛生物体的基因组中的特定目标基因座，在一些情况下需要相对适度的内切核酸酶设计、构建和应用。虽然这些技术已经使基因组工程广泛可用，但可编程核酸酶切割脱靶序列的能力可能限制其适用性并引起对治疗安全性的担忧。在这篇文章中，我们审查的方法，以评估和提高DNA切割活性的可编程位点特异性核酸内切酶和描述的程序，一个全面的脱靶分析方法的基础上，在体外选择非常大的（~1012元）库的潜在的核酸酶底物。
The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this article we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~1012-membered) libraries of potential nuclease substrates.