Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Res. 2011 Jan;39(1):359-72. doi: 10.1093/nar/gkq704. Epub 2010 Aug 10.
DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 bp sequence for AvrXa7 and 24 bp for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.
DNA 双链断裂增强了细胞内同源重组的效率，并通过利用工程化内切酶，被用于靶向基因组编辑。在此，我们报道了一组新型的、定点切割的 DNA 核酸酶的构建和初步特征，这些核酸酶由限制酶 FokI 内切酶结构域（FN）与 AvrXa7 和 PthXo1 的高特异性 DNA 结合结构域融合而成。AvrXa7 和 PthXo1 是转录激活因子样（TAL）效应子家族的成员，其中心重复单元决定了靶 DNA 的识别，并可通过模块化构建来创造新的 DNA 特异性。融合蛋白 FN-AvrXa7、AvrXa7-FN 和 PthXo1-FN 保留了其靶 DNA 的识别特异性（AvrXa7 为 26bp，PthXo1 为 24bp）和 FokI 的双链 DNA 切割活性，因此被称为 TAL 核酸酶（TALNs）。在最佳条件下，所有三种 TALNs 都能在 TAL 结合位点附近切割 DNA。当在酵母中表达时，TALNs 促进含有成对的 AvrXa7 或不对称的 AvrXa7/PthXo1 靶序列的 LacZ 基因的 DNA 同源重组。我们的结果证明了构建一个新型 TALN 工具盒的可行性，该工具盒有可能在缺乏靶向基因敲除和同源重组的机制的生物体中进行靶向基因组修饰。
