Icon Genetics GmbH, Halle/Saale, Germany.
PLoS One. 2011;6(5):e19722. doi: 10.1371/journal.pone.0019722. Epub 2011 May 19.
Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
生成针对复杂基因组中独特序列的定制 DNA 结合域对于许多生物技术应用至关重要。最近描述的黄单胞菌转录激活样效应物（TALEs）的 DNA 结合域由一系列串联排列的重复序列组成，每个重复序列结合靶序列的一个核苷酸。我们在此提出了一种基于含有 17.5 个重复序列的 AvrBs3 TALE 的工程化 TALE 蛋白的策略，作为支架，具有新型的 DNA 结合特异性。对于 17 个完整重复序列中的每一个，生成了四个模块类型，每个模块都具有不同的碱基偏好。使用这组 68 个重复模块，可以通过以定义的线性顺序组装选定的模块来构建任何 17 个核苷酸 DNA 靶序列的识别域。组装通过 Golden Gate 克隆方法在两个连续的一步式克隆步骤中进行，该方法允许多个 DNA 片段无缝融合。应用该策略，我们组装了具有新靶特异性的设计 TALEs，并在体内测试了它们的功能。
