Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.
Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.
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DOI：
10.1002/0471142727.mb1215s100
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发表时间：
2012-10
影响因子：
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通讯作者：
Sander, Jeffry D
中科院分区：
文献类型：
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作者：
Reyon, Deepak;Khayter, Cyd;Regan, Maureen R;Joung, J Keith;Sander, Jeffry D
工程化的转录激活因子样效应物核酸酶（TALEN）是用于在多种生物体和细胞类型（包括植物、斑马鱼、C.线虫、大鼠、人类体细胞和人类多能干细胞。在这里，我们描述了一个详细的协议，串行，层次组装的TALEN，既不需要PCR，也不需要专门的多片段连接，可以由任何实验室实施。这种限制性酶和连接（真实的）方案可以使用小质粒文库和用户友好的基于网络的软件来实施，该软件既鉴定感兴趣的序列中的靶位点，又生成促进TALEN组装的可打印的图形指南。进行真实的组装方法所需的所有质粒均可通过Addgene质粒分销服务（http：//www.addgene.org/talengineering）公开获得。或者，为了减少所需的时间和精力，用户可以使用编码预组装的TALE重复序列的质粒文库来练习真实的。有了我们描述的试剂、方案和软件平台，研究人员可以使用标准克隆技术在两周或更短的时间内轻松设计多个TALEN。
Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe a detailed protocol for the serial, hierarchical assembly of TALENs that requires neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. This restriction enzyme and ligation (REAL) protocol can be practiced using a small plasmid library and user-friendly, web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. All plasmids required to perform the REAL assembly method are publicly available through the Addgene plasmid distribution service (http://www.addgene.org/talengineering). Alternatively, to decrease the time and effort required, users can practice REAL using a library of plasmids encoding pre-assembled TALE repeats. With the platform of reagents, protocols, and software we describe, researchers can easily engineer multiple TALENs within two weeks or less using standard cloning techniques.