Engineering locus-specific binders to DNA modifications

项目摘要 对基因组DNA的化学修饰在所有生命王国中无处不在。它们的性质是不同的 包括核酸残基的甲基化、羟甲基化和甲酰化。这一点也越来越清楚 这些修改具有不同的监管作用。在真核生物中，DNA修饰调节基因表达， RNA剪接、基因组组织和基因印记；这些分子功能也具有重要的作用 发展、疾病和生物与环境刺激的相互作用的下游后果 化学制品。此外，随着基因组和表观基因组编辑技术的出现，这些修改可以 偏向于分子工具的特异性和有效性。鉴于DNA修饰的广泛重要性，任何 能够控制、测量、感知或跟踪它们的实验工具将对 生物学科。特别需要的，但在技术上还不可能的是，有能力 同时检测活细胞和单细胞中特定基因组位置的表观遗传修饰。这 这一能力将开启一系列新的实验方法，以揭示对以下主题的新见解 基因印记，表观遗传修饰的因果关系和相关性，以及细胞和组织的随机性。 此外，它还将为新型亲和试剂的开发提供信息，用于生化分析、疾病 检测和诊断以及基因组和表观基因组编辑技术 进入DNA修饰状态。为了推动这一新的研究领域，我们将创造第一个分子工具 在特定和可编程的基因组位置结合DNA羟甲基化，并提供广泛适用的 为设计许多其他类似的分子试剂提供了平台。

Project Summary Chemical modifications to genomic DNA are ubiquitous across all kingdoms of life. They are diverse in nature including methylation, hydroxymethylation, and formylation of nucleic acid residues. It is also increasingly clear that these modifications have diverse regulatory roles. In eukaryotes, DNA modifications regulate gene expression, RNA splicing, genome organization, and gene imprinting; these molecular functions also have important downstream consequences for development, disease, and organismal interactions with environmental stimuli and chemicals. Furthermore, with the advent of genome and epigenome editing technologies, these modifications can bias the specificity and efficiency of molecular tools. Given the broad importance of DNA modifications, any experimental tools that are able to control, measure, sense, or track them would have profound impacts across biological disciplines. What is particularly needed, but not yet technologically possibly, is the ability to simultaneously sense epigenetic modifications at specific genomic locations in living and single cells. This capability would unlock a broad palette of new experimental approaches to reveal new insights into topics such as gene imprinting, causation vs. correlation of epigenetic modifications, and cell and tissue stochasticity. Furthermore, it would inform the development of new classes of affinity reagents for biochemical assays, disease detection, and diagnostics and of genome and epigenome editing technologies that are aware of and specific towards DNA modification state. To drive this new realm of studies, we will create the first molecular tool that binds DNA hydroxymethylation at specific and programmable genomic loci, and provide a broadly applicable platform for engineering many other similar molecular reagents.