Li Ting, Liu Bo, Chen Chih Ying, Yang Bing
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, USA.
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, USA.
Methods. 2014 Aug 15;69(1):9-16. doi: 10.1016/j.ymeth.2014.03.019. Epub 2014 Mar 27.
Transcription activator-like effector nucleases (TALENs), the newly developed and powerful genetic tools for precise genome editing, are fusion proteins of TAL effectors as DNA binding domains and the cleavage domain of FokI endonuclease. As a pair, the central repeat regions of TALENs determine the DNA binding specificity for the two sub-target sites; and the dimeric non-specific FokI cleavage domains cause a DNA double strand break (DSB) between the bound sequences. In vivo, cells repair the DSBs through either non-homologous end joining (NHEJ) pathway or homologous recombination (HR) pathway. Various methods have been developed for easy and fast assembly of TALEN genes for their utilization in a variety of eukaryotic cells or organisms. Here we present a TALEN-based rice genome modification protocol including constructing modularly assembled TALENs, rice transformation, and mutant screening.
转录激活样效应因子核酸酶（TALENs）是新开发的用于精确基因组编辑的强大基因工具，它是作为DNA结合结构域的TAL效应因子与FokI核酸内切酶的切割结构域的融合蛋白。作为一对，TALENs的中央重复区域决定了对两个亚靶位点的DNA结合特异性；而二聚化的非特异性FokI切割结构域会在结合序列之间造成DNA双链断裂（DSB）。在体内，细胞通过非同源末端连接（NHEJ）途径或同源重组（HR）途径修复DSB。已经开发出各种方法，以便轻松快速地组装TALEN基因，用于多种真核细胞或生物体。在此，我们展示了一种基于TALEN的水稻基因组修饰方案，包括构建模块化组装的TALENs、水稻转化和突变体筛选。
