Key Laboratory of Horticulture Science for Southern Mountainous Regions, Southwest University, Chongqing, 400715, China.
J Integr Plant Biol. 2013 Nov;55(11):1092-103. doi: 10.1111/jipb.12091. Epub 2013 Sep 18.
Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.
具有 DNA 核酸酶的位点特异性识别模块作为基因组靶向的分子工具具有巨大的潜力。III 型转录激活子样效应物（TALEs）包含一个由串联重复组成的 DNA 结合结构域，可通过工程设计使其与用户定义的特定 DNA 序列结合。我们证明了使用称为“单元组装”的方法构建的定制 TALE 核酸酶（TALENs）可特异性靶向 Brassica oleracea L. var. capitata L. 中的内源 FRIGIDA 基因。结果表明，TALENs 在体外和体内与靶位点结合并切割双链 DNA，而效应物结合元件具有 23 bp 的间隔。T7 内切酶 I 测定和测序数据表明，TALENs 产生了双链断裂，这些断裂通过靶序列内的非同源末端连接途径进行修复。这些数据表明，定制 TALENs 可应用于靶向和修饰基因组，在那些缺乏基因靶向方法的生物体中进行缺失，以提供在育种改良中进行种质资源。
