Analysis of TALE-DNA Recognition

描述（由申请人提供）：转录激活因子样效应物（TALEs）的DNA结合域（DBDs）包含重复变量双残基（RVD）结构域的模块化组装，最近发现其具有非常简单的DNA识别代码，其中单个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一个有吸引力的选择，以创建定制的TALE核酸酶（TALENs），用于在特定的目标基因组位点上创建突变，用于与转录激活域融合以靶向激活基因表达，转录抑制域融合以靶向抑制基因表达，与羟化酶催化域融合以靶向CpG去甲基化。或者是组蛋白去甲基化酶用于靶向赖氨酸特异性去甲基化。然而，对TALE-DNA识别特性的深入分析尚未进行。潜在的脱靶特异性可能导致突变，替代识别位点和/或由TALENs引起的毒性增加，或由人工TALE tf引起的基因表达的不希望的改变或表观遗传修饰。在这个项目中，我们将研究TALE-DNA识别中潜在的上下文依赖效应。此外，我们将研究TALE重复序列在TALE DBD内位置的潜在影响，以及目标序列两侧的周围序列组成和DNA形状对TALE结合的潜在影响。我们还将检测从分子模型中预测的具有改进的DNA结合特异性的重新设计的TALEs。任何此类上下文依赖性都可能导致脱靶特异性和/或改变结合效率，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TALE蛋白的项目中加以考虑。我们将使用结果数据生成TALE DNA结合特异性的计算模型，包括实现用于预测用户输入TALE的DNA结合特异性的在线工具，以及为用户输入感兴趣的DNA序列区域设计TALE蛋白质。我们还将比较体外TALE-DNA结合数据与体内靶特异性。

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.