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.
