EAGER: Site-specific engineering of DNA methylation states in plant genomes

该项目将设计植物基因组的化学特征，作为理解这些特征如何控制植物性状的前奏。在植物基因组中，许多胞嘧啶残基通过添加甲基进行化学修饰。DNA甲基化的存在通常与基因表达的变化相关，但这种变化如何影响特定性状尚不清楚。本研究将开发和实施在拟南芥基因组特定位点添加或去除DNA甲基化的方法，从而为分析DNA甲基化对基因功能的影响铺平道路。研究目标将与高中生和研究生合作实现，他们将获得最先进的基因组工程技术的宝贵经验。这种技术提供了一种在不改变DNA序列的情况下控制基因表达状态的方法，可以在基础和应用植物生物学中产生广泛的影响。基因组的表观遗传修饰，如DNA中胞嘧啶的甲基化，被广泛认为与表型性状的变化有关。然而，目前还没有系统的方法来直接检验这些观察结果的因果关系。本项目旨在开发一套通用工具，用于植物基因组DNA甲基化状态的位点特异性工程。为了调节基因组特定区域的DNA甲基化状态，控制DNA甲基化的酶将使用CRISPR-dCas9系统连接到感兴趣的靶标上。该方法将首先在酵母（Saccharomyces cerevisiae）中开发，因为它具有小的，未甲基化的基因组，这将允许轻松评估甲基化状态的靶向和脱靶变化。一旦对酵母进行了优化，这些方法将在模式植物拟南芥中应用，以靶向位点特异性甲基化和去甲基化。通过这项研究开发的工具将使测试有关不同甲基化状态和表型的假设成为可能，从而使“反向表观遗传学”方法能够用于基因功能和基因发现的预测研究。该奖项由生物科学理事会分子和细胞生物科学部的遗传机制和系统与合成生物学项目共同资助。

This project will engineer chemical features of plant genomes as a prelude to understanding how those features control plant traits. In plant genomes, many cytosine residues are chemically modified by addition of methyl groups. The presence of DNA methylation is often correlated with changes in gene expression, but how such changes influence specific traits is not well understood. This research will develop and implement methods for adding or removing DNA methylation at specific sites in the Arabidopsis thaliana genome, thereby paving the way for analyzing the effects of DNA methylation on gene function. Research goals will be achieved in collaboration with high school and graduate students, who will obtain valuable experience in state-of-the-art genome engineering technology. This kind of technology could have broad impact in both basic and applied plant biology by providing a way to control gene expression states without changing the DNA sequence.Epigenetic modifications to genomes, such as methylation of cytosines in DNA, are widely known to be associated with changes in phenotypic traits. However, there is currently no systematic method to directly test the causality of these observations. This project aims to develop a set of universal tools for site-specific engineering of DNA methylation states in plant genomes. To modulate DNA methylation states at specific regions of the genome, enzymes that control DNA methylation will be tethered to targets of interest using the CRISPR-dCas9 system. The approach will first be developed in the yeast, Saccharomyces cerevisiae, as it has a small, unmethylated genome, which will permit easy evaluation of both on- and off-target changes in methylation states. Once optimized for yeast, the methods will be deployed in the model plant, Arabidopsis thaliana, to target site-specific methylation and de-methylation. The tools developed through this research will make it possible to test hypotheses relating differential methylation states and phenotypes to enable "reverse epigenetics" approaches for predictive studies of gene function and for gene discovery. This award was co-funded by the Genetic Mechanisms and the Systems and Synthetic Biology Programs in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate.