Analysis of TALE-DNA Recognition

描述(由申请人提供)：转录激活物样效应器(TALE)的DNA结合结构域(DBD)包含重复可变双残基(RVD)结构域的模块化组件，最近发现其具有极其简单的DNA识别码，由此单个RVD识别单个核苷酸。这种简单的模块化使定制设计的TALE蛋白成为与核酸酶融合的诱人选择，用于在特定的目标基因组位点产生突变，融合到转录激活域以定向激活基因表达，融合到转录抑制域用于靶向抑制基因表达，融合到羟基酶催化域用于靶向CpG去甲基化，或组蛋白去甲基化用于靶向赖氨酸特异性去甲基化。然而，对TALL-DNA识别特性的深入分析还没有进行。潜在的非靶标特异性可能导致突变和/或由TALEN引起的替代识别位点和/或增加的毒性，或由人工故事TF引起的基因表达的不良改变或表观遗传修饰。在这个项目中，我们将研究TALL-DNA识别中潜在的上下文相关效应。此外，我们还将研究TALL重复序列在TALL DBD内的位置的潜在影响，以及周围序列组成和靶序列两侧DNA形状对TALL结合的潜在影响。我们还将分析从分子建模预测的重新设计的故事，以提高DNA结合的特异性。任何这样的上下文依赖都可能导致脱靶特异性和/或结合效率的改变，并且需要在旨在设计用于基因组工程或其他合成生物学应用的定制TALL蛋白的项目中被考虑。我们将使用得到的数据来生成TALL DNA结合特异性的计算模型，包括实施在线工具来预测用户输入的TALL的DNA结合特异性，并为用户输入的感兴趣的DNA序列区域设计TALE蛋白。我们还将比较体外TALL-DNA结合数据和体内靶标的特异性。

DESCRIPTION (provided by applicant): The DNA binding domains (DBDs) of transcription activator-like effectors (TALEs) contain modular assemblies of Repeat Variable Diresidue (RVD) domains, which were recently discovered to have a strikingly simple DNA recognition code, whereby an individual RVD recognizes a single nucleotide. This simple modularity has made custom- designed TALE proteins an attractive option for fusion to nucleases to create customized TALE nucleases (TALENs) for use in creating mutations at specific, target genomic sites, for fusion to a transcriptional activation domain for targeted activation of gene expression to a transcriptional repression domain for targeted repression of gene expression, to a hydroxylase catalytic domain for targeted CpG demethylation, or to a histone demethylase for targeted lysine-specific demethylation. However, in-depth analysis of TALE-DNA recognition properties has not been undertaken. Potential off- target specificities could result in mutations a alternate recognition sites and/or increased toxicity resulting from TALENs, or undesired alterations of gene expression or epigenetic modifications resulting from artificial TALE TFs. In this project, we will investigate potential context-dependent effects in TALE-DNA recognition. In addition, we will investigate the potential impact of the position of TALE repeats within the TALE DBD, and also the potential influence of the surrounding sequence composition and DNA shape flanking the target sequence on TALE binding. We will also assay re-engineered TALEs predicted from molecular modeling to have improved DNA binding specificities. Any such context dependencies could result in off- target specificities and/or altered efficiencies of binding, and would need to be considered in projects that aim to design custom TALE proteins for use in genome engineering or other synthetic biology applications. We will use the resulting data to generate a computational model of TALE DNA-binding specificity, including implementation of online tools for predicting the DNA binding specificities of user-input TALEs and for design of TALE proteins for user-input DNA sequence regions of interest. We will also compare in vitro TALE-DNA binding data to in vivo target specificities.