Cermak Tomas, Starker Colby G, Voytas Daniel F
Department of Genetics, Cell Biology & Development, University of Minnesota, 350 Cargill Building, 1500 Gortner Avenue, St. Paul, MN, 55108-1023, USA.
Methods Mol Biol. 2015;1239:133-59. doi: 10.1007/978-1-4939-1862-1_7.
An important breakthrough in the field of genome engineering was the discovery of the modular Transcription Activator-Like Effector (TALE) DNA binding domain and the development of TALE nucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species. Underlying the boom in genome engineering is the striking simplicity and low cost of engineering new specificities of TALENs and other sequence-specific nucleases. In this chapter, we describe a rapid, inexpensive, and user-friendly protocol for custom TALEN construction based on one of the most popular TALEN assembly platforms, the Golden Gate cloning method. Using this protocol, ready-to-use TALENs with specificity for targets 13-32 bp long are constructed within 5 days.
基因组工程领域的一项重要突破是模块化转录激活样效应物（TALE）DNA结合结构域的发现以及TALE核酸酶（TALENs）的开发。TALENs使研究人员能够在目标位点产生DNA双链断裂，以创建基因敲除或引入特定的DNA序列修饰。精确的基因组工程越来越多地用于研究基因功能、开发疾病模型或在作物物种中创造新性状。TALENs和其他序列特异性核酸酶工程新特异性的显著简单性和低成本是基因组工程蓬勃发展的基础。在本章中，我们基于最流行的TALEN组装平台之一——金门克隆方法，描述了一种快速、廉价且用户友好的定制TALEN构建方案。使用该方案，可在5天内构建出对13 - 32 bp长的靶标具有特异性的即用型TALENs。
