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Abstract
Crop improvement through traditional plant breeding has paved the way for a healthier and more productive society. Despite improvements to crop yield and nutritional value, food security remains one of society's most urgent and challenging problems. Therefore, improvements to the current technologies, such as CRISPR-Cas9 genome engineering, are necessary to meet the global food demands. Although the CRISPR gene editing system has been implemented into many research or plant breeding programs, the ability to quickly engineer a specific trait of interest can still require substantial time, resources, and effort. These challenges can be attributed, in part, to the incomplete understanding of the rules that govern CRISPR-Cas9 genome editing efficacy. To realize the full potential of gene editing, new strategies to increase multiplexed editing efficiency and precision are needed.
In the following three chapters I use the model organisms Setaria viridis and Arabidopsis thaliana to address these challenges. In Chapter I, I describe the development of a robust multiplexed editing and transformation pipeline for the emerging monocot model, Setaria viridis. To achieve the efficient creation of genetically modified plants, a protoplast system was used to rapidly test and optimize gene editing reagents. These optimized CRISPR-Cas9 editing reagents, capable of improved multiplexed genome editing, were then transformed into S. viridis using the standard tissue culture method. Highly efficient transformation enabled the creation of transgene-free plants harboring frameshift mutations at the target genes.
In Chapter II, I leverage our improved Setaria viridis gene editing pipeline to create a panel of mutants with perturbed DNA methylomes. To accomplish this, we designed, built, and transformed T-DNA constructs to individually target four different DNA methylation families: METHYLTRANSFERASE1 (MET1), DECREASE IN DNA METHYLATION1 (DDM1), CHROMOMETHYLASE (CMT), and DOMAINS REARRANGED METHYLTRANSFERASE (DRM). Currently, the DRM1 mutant has been successfully isolated. Chapter II focuses on the phenotypic and molecular characterization of this mutant.
In Chapter III, I report a series of experiments to better understand the rules that govern CRISPR-Cas9 genome editing. Two features have been implicated in CRISPR-Cas9 editing efficacy: (1) the DNA sequence of the CRISPR-Cas9 target site, and (2) nonsequence features such as epigenetic context at the CRISPR-Cas9 target site. Previous studies have suggested that the DNA sequence being targeted for gene editing is the primary determinant of CRISPR-Cas9 efficiency and precision. As such, several tools have been developed to predict gene editing efficiency and precision solely based on the gene’s DNA sequence. However, the reliability of these tools varies and appears to translate poorly to plants. This observation strongly suggests that nonsequence factors, such as epigenetic context, could influence CRISPR gene editing in plants. In fact, this possibility has recently been demonstrated; however, these studies were limited by their inability to separate the effects of the gene’s DNA sequence from its epigenetic features. To address the challenge of separating these two variables, we developed the strategy of editing identical DNA sequences that are located in different epigenetic contexts. This strategy enabled me to systematically dissect the influences of nonsequence features on CRISPR-Cas9 efficacy, revealing the significant influence of distinct epigenetic features.
