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

植物基因组调控区的工程设计有可能促进确保粮食安全所需的快速作物改良。这个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--并建立工具来设计调控DNA，用于作物改良。基因表达受顺式调控元件控制，包括启动子、增强子、沉默子和绝缘子。工程改造这些元件是改善作物性状的一种有前途的策略。调节区的变化通常会导致基因表达的组织或条件特异性变化，因此可能比编码变化危害更小。重要的是，进化和驯化都经常作用于调控DNA。然而，我们仍然缺乏足够的知识，顺式调控元件和他们的语法，使常规的工程这些元素的作物改良。我最近开发了植物STARR-seq，这是一种大规模平行报告分析，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来进一步理解植物基因调控：（1）我将表征植物绝缘子和沉默子，因为这些元件是紧密控制转基因表达以实现有效作物工程所必需的;（2）我将研究顺式调控元件内部和之间的相互作用;（3）研究顺式调控元件和反式作用蛋白之间的高阶相互作用是如何微调植物基因表达的。这些研究将使我们更深入地了解植物调控DNA的活性和强度的规则，并使我们能够准确地预测和操纵植物基因表达。在研究植物调控语法的同时，我将建立一个顺式调控元件的无转基因组编辑工具包。目前植物基因组定向工程的技术解决方案面临公众对转基因生物的怀疑和对脱靶效应的担忧。在这个项目中，我将开发一种替代方法来植物基因组工程，通过使用编辑蛋白质，这些蛋白质在农杆菌中表达，随后转移到植物细胞中，而不伴随DNA的转移。该系统将产生无转基因植物并减少脱靶突变的数量，因为编辑蛋白质不会组成性产生。

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.