Learning the grammar of plant regulatory DNA for targeted engineering of gene expression

植物基因组调控区域的工程有可能促进确保粮食安全所需的作物快速改良。该项目将分析植物顺式调控元件，以确定它们相互作用的方式--调控语法--并建立工具，以设计用于作物改良的调控DNA。基因表达受启动子、增强子、抑制子和绝缘体等顺式调控元件的调控。设计这些元素是一种很有前途的改良作物性状的策略。调控区域的变化通常会导致组织或条件特定的基因表达变化，因此比编码变化的危害要小。重要的是，进化和驯化都频繁地作用于调控DNA。然而，我们仍然缺乏足够的关于顺式调控元件及其语法的知识，无法使这些元件的常规工程用于作物改良。我最近开发了一种植物STARR-SEQ，一种大规模平行的报告分析，可以表征顺式调控元件的活性。在这个项目中，我将使用植物STARR-SEQ来加深我们对植物基因调控的理解：(1)我将表征植物绝缘体和消音器，因为这些元件是有效作物工程中严格控制转基因表达所必需的；(2)我将研究顺式调控元件内部和之间的相互作用；以及(3)我将研究顺式调控元件和反式作用蛋白之间的高阶相互作用如何微调植物基因的表达。这些研究将使我们更深入地了解植物调控DNA的活性和强度的规律，并使我们能够准确地预测和操纵植物的基因表达。在学习植物调控语法的同时，我将建立一个工具包，用于对顺式调控元件进行无转基因基因组编辑。目前植物基因组定向工程的技术解决方案面临着公众对转基因生物的怀疑和对非靶标效应的担忧。在这个项目中，我将开发一种替代的植物基因组工程方法，通过编辑在农杆菌中表达的蛋白质，然后转移到植物细胞中，而不伴随DNA的转移。这个系统将产生非转基因植物，并减少非靶标突变的数量，因为编辑蛋白将不会被结构性地产生。

Engineering of regulatory regions in plant genomes has the potential to facilitate rapid crop improvements necessary for ensuring food security. This project will analyze plant cis-regulatory elements to determine the ways in which they interact—the regulatory grammar—and build tools to engineer regulatory DNA for crop improvement. Gene expression is controlled by cis-regulatory elements that include promoters, enhancers, silencers and insulators. Engineering such elements is a promising strategy to improve crop traits. Changes in regulatory regions often cause tissue- or condition-specific changes in gene expression, and hence can be less deleterious than coding changes. Importantly, evolution and domestication both have frequently acted on regulatory DNA. However, we still lack sufficient knowledge of cis-regulatory elements and their grammar to make routine the engineering of these elements for crop improvement. I recently developed plant STARR-seq, a massively parallel reporter assay that can characterize the activity of cis-regulatory elements. In this project, I will use plant STARR-seq to further our understanding of plant gene regulation: (1) I will characterize plant insulators and silencers, as these elements are needed to tightly control the expression of transgenes for effective crop engineering; (2) I will study the interactions within and between cis-regulatory elements; and (3) I will study how higher-order interactions between cis-regulatory elements and trans-acting proteins fine-tune plant gene expression. These studies will lead to a deeper understanding of the rules that govern the activity and strength of plant regulatory DNA and will enable us to accurately predict and manipulate plant gene expression. In parallel to studying plant regulatory grammar, I will establish a toolkit for transgene-free genome editing of cis-regulatory elements. Current technical solutions for targeted engineering of plant genomes face public skepticism about transgenic organisms and concerns around off-target effects. In this project, I will develop an alternative approach to plant genome engineering by using editing proteins that are expressed in Agrobacteria and subsequently transferred to plant cells without a concomitant transfer of DNA. This system will yield transgene-free plants and reduce the number of off-target mutations since the editing proteins will not be produced constitutively.