| Genome editing is an exciting technology that enables scientists to introduce precise genetic modifications, using engineered DNA-binding proteins that can be "programmed" to target specific genomic sequences, and is often partnered with an additional effector domain to form artificial transcription factors (ATFs) or nucleases. Several protein platforms have been proposed, each with their own strengths and weaknesses, as well as ease of use. However, no platform is perfectly specific, and off-target binding events remain a central concern for users of these technologies-particularly those with interests in therapeutics and clinical settings. This body of work encompasses two major projects across three chapters, the first being the use of ATFs for use in Plasmodium falciparum, the causative agent of malaria, a disease that affects over 100 million people annually and continues to evade elimination efforts in endemic nations. We propose a novel tool using TALE-based artificial transcription factors, transported into the parasite nucleus via cell-penetrating peptide, to modify the expression of endogenous parasite genes. The second project, split into two chapters, focuses on the development of bioinformatics software tools to process high-throughput sequencing and epigenetic data in order to investigate the binding specificity of three popular genome editing platforms: zinc fingers, TALEs, and CRISPR/Cas9. Using these tools, we were able to corroborate biological observations of an N-terminal/5' polarity in TALE binding and discover a novel binding motif for the endogenous transcription factor Miz-1. However, CRISPR/Cas9 specificity with regard to local chromatin structure remains unclear. We also investigated the ability CRISPR/Cas9 to interact with DNA in ostensibly located in closed chromatin regions and discovered that no single chromatin marker appears to be a sufficient predictor of Cas9-mediated cleavage.;The methodologies developed in this dissertation are not limited to engineered proteins, and could be used to explore the binding behavior of other DNA-binding moities, such as endogenous transcription factors. As a whole, this dissertation seeks to understand the underlying biology of engineered DNA-binding proteins, in hopes of improving genome editing technologies, and applying our knowledge of these platforms for use in new systems. |
