Maeder Morgan L, Thibodeau-Beganny Stacey, Sander Jeffry D, Voytas Daniel F, Joung J Keith
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Nat Protoc. 2009;4(10):1471-501. doi: 10.1038/nprot.2009.98. Epub 2009 Sep 17.
Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN selections are carried out in Escherichia coli using a bacterial two-hybrid system and do not require specialized equipment. Here we provide a detailed protocol for carrying out OPEN to engineer zinc-finger arrays that have a high probability of functioning as ZFNs. Using OPEN, researchers can generate multiple, customized ZFNs in approximately 8 weeks.
工程化锌指核酸酶(ZFNs)构成了一种广泛适用的方法的基础,用于对真核基因组进行靶向、高效修饰。在最近的工作中,我们描述了OPEN(寡聚体库工程),这是一种基于组合选择的“开源”方法,用于构建能作为ZFNs发挥良好功能的锌指阵列。我们还通过直接比较表明,OPEN方法在构建ZFNs方面比先前描述的“模块化组装”方法具有更高的成功率。OPEN筛选在大肠杆菌中使用细菌双杂交系统进行,并且不需要专门的设备。在这里,我们提供了一个详细的方案,用于实施OPEN以构建极有可能作为ZFNs发挥功能的锌指阵列。使用OPEN,研究人员可以在大约8周内生成多个定制的ZFNs。