CAREER: Tailoring hormone responses in plants via synthetic signal integration devices

植物激素生长素是一种重要的植物生长调节剂，它调控植物生长和生理的多个方面，并在植物生活的每个阶段发挥作用。该项目的目标是产生新的分子工具，以精确控制生长素在哪里以及何时产生，作为一种控制植物生长的方法，特别是在植物的根部。利用强大的基因编辑和靶向技术(CRISPR)，该项目生成了可编程的遗传设备(生物逻辑门)，使其能够随意打开和关闭感兴趣的基因(如生长素生物合成基因)。在该项目中开发的基本原理和工具具有在农业和其他领域广泛应用的潜力。在这项研究中产生的方案和材料作为北卡罗来纳州立大学一门新的合成生物学课程的基础，该课程面向北卡罗来纳州立大学的高级本科生和初级研究生。此外，调查员还将其中一些材料改编成讲习班，为高中教师提供培训和资源。研究人员还建立了一个分子生物学工具“借阅图书馆”，使当地教师能够在自己的教室里进行分子实验。此外，在北卡罗来纳州自然科学博物馆和当地小学继续之前建立的双语(英语和西班牙语)动手Plants4Kids演示，用活的植物和种子为最年轻的观众带来实验科学的兴奋。这些活动使年轻人接触到科学方法并揭开农业和植物基因技术的神秘面纱。生物学研究往往受到可用启动子的选择的限制，这些启动子可以以复杂的模式和所需的水平驱动感兴趣的基因的表达。为了克服这一限制，开发了一系列基于CRISPR的易于编程的合成遗传电路，以整合来自先前描述的合成和自然启动子的多个输入。这些新工具利用crRNA：trrRNA gRNA对，并结合来自两个或更多不同驱动/启动子的输入来划定基于dCas9的合成转录因子激活或抑制目标基因表达的位置。该方法依赖于两个或多个伪正交gRNA对，并且与所有基本逻辑门的构建兼容，以产生目标基因表达的多种派生模式。从理论上讲，这项技术可以产生数百个组合在一起执行复杂逻辑运算的正交遗传组件。新的策略不仅是可扩展的，而且能够调节基因表达水平，从而在靶向基因表达方面提供了前所未有的灵活性。为了证明新方法的有效性，研究人员调节了生长素缺乏突变体的根中局部生长素生产的组织特异性表达，并询问了对根构型的影响；从而解决了长期存在的关于本地生长素生产在维持干细胞生态位或植物根方面的作用的问题。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The plant hormone, auxin is an important plant-made growth regulator that governs multiple aspects of plant growth and physiology and acts at every stage in the life of the plant. The goal of this project is to generate new molecular tools to precisely control where and when auxin is made as a way to manipulate plant growth, particularly in the root. Taking advantage of the powerful gene editing and targeting technology (CRISPR), this project generates programmable genetic devices (biological logic gates), that make it possible to turn genes of interest (such as auxin biosynthesis genes) on and off at will. The basic principles and tools developed in this project have the potential for broad applications in agriculture and beyond. The protocols and materials generated in this study serve as the basis for a new Synthetic Biology course for upper level undergraduate and beginning graduate students at NC State University. In addition, the investigator has adapted some of these materials for workshops that provide training and resources for high school teachers. The investigator also establishes a molecular biology tool "lending library" that enables local teachers to execute molecular experiments in their classrooms. Furthermore, continuation of the previously established bilingual (English and Spanish) hands-on Plants4Kids demonstrations with live plants and seeds at the NC Museum of Natural Sciences and local elementary schools brings the excitement of experimental sciences to the youngest audiences. These activities expose young people to scientific methods and demystify agricultural and plant genetic techniques.Biology research is often restricted by the choice of promoters available to drive the expression of genes of interest in complex patterns and at desired levels. To overcome this limitation, a series of easily programmable CRISPR-based synthetic genetic circuits are developed to integrate multiple inputs from previously described synthetic and natural promoters. These new tools take advantage of crRNA:tracrRNA gRNA pairs and combine inputs from two or more different drivers/promoters to delimit where a dCas9-based synthetic transcription factors activate or represses target gene expression. The proposed approach rests on two or more pseudo-orthogonal gRNA pairs and is compatible with the construction of all basic logic gates to produce multiple derived patterns of targeted gene expression. In theory, this technology can produce hundreds of orthogonal genetic components that in combination perform complex logic operations. The new strategy is not only scalable, but also enables tuning of gene expression levels, thus providing an unprecedented degree of flexibility in targeted gene expression. To demonstrate the utility of the new approach, the investigator regulates the tissue specific expression of local auxin production in roots in an auxin-deficient mutant and interrogates the effects on root architecture; thus addressing a long-standing question on the role of local auxin production in maintaining the stem cell niche or plant roots.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.