Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors
Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors
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DOI：
10.1073/pnas.1013133107
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发表时间：
2010-12-14
影响因子：
11.1
通讯作者：
Lahaye, Thomas
中科院分区：
文献类型：
--
作者：
Morbitzer, Robert;Roemer, Patrick;Lahaye, Thomas
可以量身定制以结合所需DNA序列的蛋白质是分子生物学的关键工具。先前的研究表明，来自细菌属的xanthomonas的转录激活效应子（TALES）的DNA结合特异性是由串联34/35-氨基酸重复单位的反复变量的Diresidues（RVD）定义的。我们研究了两个故事的嵌合体，这些故事在RVD和非RVD上有所不同，发现与RVD的关键贡献相反，非RVD对嵌合体的DNA结合特异性没有重大影响。这一发现表明，只需要修改RVD即可生成设计器故事（DTALES）以激活用户定义的目标基因的转录。我们使用了Tale Avrbs3的脚手架，并更改了RVD，以匹配番茄BS4，拟南芥EGL3或拟南芥KNAT1启动子。所有三个dtales都以序列特异性的方式转录激活了所需的启动子，因为靶向DNA序列中的突变消除了启动子的激活。这项研究是独一无二的，表明染色体基因座可以由Dtales专门针对。我们还设计了两个AVRBS3衍生物，其中有四个额外的重复单元专门激活Pepper BS3或UPA20启动子。由于AVRBS3激活两个启动子，因此我们的数据表明，添加重复单元有助于特定于故事的微调。最后，我们证明了RVD NK介导了与格努德替氏菌的特定相互作用，到目前为止，这些相互作用无法通过任何已知的RVD类型专门针对。总而言之，我们的数据表明，可以根据整个基因组中的用户定义的DNA序列量身定制这个故事支架。
Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of user-defined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated the desired promoters in a sequence-specific manner as mutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NK mediates specific interaction with Gnucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.