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Abstract
An understanding of gene expression is essential to elucidate how altered phenotypes arise in eukaryotic organisms. Importantly, modifications not directly altering the DNA sequence of genes can alter gene expression in profound ways and can result in the emergence of novel phenotypes. Such modifications often are caused by changes to the epigenome, or through the alteration of cis-regulatory elements (CREs). DNA methylation, a modification to the DNA base cytosine, is a primary component of the epigenome, and can serve to alter gene expression of genes, particularly when found in promoter sequences. Alterations to cis-regulatory elements can result in altered transcription factor binding at the promoters of target genes, resulting in altered expression patterns and the emergence of novel phenotypes.My PhD research focuses on creating new methods to intentionally alter cis-regulatory elements and DNA methylation, with the goal of creating novel phenotypes from otherwise genetically identical individuals. To this end, I was the lead molecular biologist for the development of epimutagenesis, a method whereby the expression of a mammalian demethylase protein in Arabidopsis thaliana stochastically removes DNA methylation throughout the genome, and results in the emergence of novel phenotypes, some of which are stably inherited over generational time. Next, I sought to create a simplified method for creating genetic variation at CREs by leveraging the multiplexing ability of Cas12a proteins. To this end, I created a heat- shock protocol for the efficient transmission of mutated alleles through the germline of A. thaliana, plants and successfully applied this method for multiplex engineering of a CRE with altered DNA sequences. Finally, I sought to leverage the development of new CRISPR RNA- editing technology for the application of silencer element detection in a genome-wide fashion. Taken together, my research has resulted in the establishment and successful application of two methods for both DNA demethylation and CRE editing in A. thaliana.