Zinselmeier, Matthew2023-09-192023-09-192022-05https://hdl.handle.net/11299/256986University of Minnesota Ph.D. dissertation. May 2022. Major: Biochemistry, Molecular Bio, and Biophysics. Advisors: Daniel Voytas, Michael Smanski. 1 computer file (PDF); ix, 141 pages.Agriculture is a centuries old practice that has selected upon natural variation over time. Highly productive cultivars today are the result of this selection. DNA sequencing has revealed the genetic blueprint for many of these crop species, allowing for precise selection of variants for breeding. Crops must survive and reproduce efficiently to be utilized by humans for agriculture. To accomplish this, crops put the DNA blueprint into action through gene expression, allowing for development and survival in the face of stress. Thus, understanding and controlling gene expression will be important for engineering highly productive crops around the world. In nature, transcription factors (TFs) are responsible for regulating gene expression. TFs are comprised of a DNA binding domain and transcription regulatory domain. The DNA binding domain will bind to a region in the genome, while the regulatory domains interact with other proteins capable of either initiating or blocking transcription. As programmable nuclease technology like CRISPR-Cas9 was elucidated, Programmable Transcription Activators (PTAs) were developed to function as engineered transcription factors controlling gene expression. PTAs can be targeted anywhere in the genome to activate expression of a target gene promoter. PTAs can also activate the expression of multiple genes at once. In Chapter One the status of PTA technology is reviewed, with systems showing promise in plant backgrounds given consideration. To ultimately use PTAs for basic plant research, we first set out to optimize PTAs for efficient performance across plant species. The VP64 activation domain is the most frequently used activation domains regardless of PTA system. This domain is derived from a human herpes virus yet is still used in many plant systems. To address this we designed, built, and tested a library ofplant-derived activation domains for activity in plant cells, as described in Chapter Two. The AvrXa10, Dof1, and DREB2 ADs proved to be efficient across a variety of plant species. We also demonstrate the use of the DREB2 AD to activate distal enhancers in A. thaliana protoplasts, showcasing the versatility of plant-optimized PTAs. Finally, PTAs were used to engineer a circuit for genetic biocontainment as described in Chapter 3. In certain crop species, there is a desire to prevent gene flow between closely related individuals. A system was devised called Engineered Genetic Incompatibility (EGI) to prevent crop transgenes from escaping into closely related weedy populations. To engineer this system a gene is identified that is capable of producing lethality when overexpressed using PTAs. Gene editing is then performed to remove the PTA binding site to comprise the EGI organism; a PTA is expressed alongside the modified promoter within an individual to prevent self-targeting. Upon crossing with a wild-type individual, hybrid progeny will contain one copy of the PTA and one copy of the unedited promoter resulting in lethal overexpression. Our efforts to implement EGI in the model organism A. thaliana are described in detail including selection of target genes, testing sgRNA activity, generating transgenic lines, and promoter mutagenesis. The work described in this thesis illustrates the development and application of PTA technology in agriculture to engineer more productive crops through controlling gene expression.enActivation DomainCRISPR-Cas9Plant EngineeringProgrammable Transcription ActivatorThesis or Dissertation