"Peptide Nucleic Acids and CRISPR-Cas9: Mechanisms and Rational Applica" by Nicholas George Economos

Date of Award

Spring 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Glazer, Peter

Abstract

In the past decade, gene editing technology development has exploded across life sciences fields with promise to advance research, agriculture, industry, and the development of curative human therapeutics. Initially lead by CRISPR-associated nucleases and their ability to induce targeted DNA double-strand breaks (DSBs), the gene editing toolbox continues to grow as new technologies emerge and improve. While the goal of these tools is to precisely manipulate DNA sequences in living cells, not all modifications are created equal. Small or large deletions and insertions, precise nucleotide changes, and combination approaches each entail different challenges and require cooperation with endogenous repair pathways to achieve an intended effect. No one tool can accomplish these biochemical feats in a one-fits-all manner. Thus, it is imperative to advance current technologies and create new approaches as diverse as the problems they set out to solve while maximizing safety for potential use in human therapy. The overarching goal of this dissertation is to apply principles of DNA repair and nucleic acid biochemistry to advance our understanding and implementation of gene editing tools across applications. First, using a novel high-throughput platform to detect nucleic acid structure interactomes, we reveal that synthetic triplex-forming peptide nucleic acids (PNAs) bind DNA strands to elicit repair factors and pathways previously unknown to participate in site-directed gene editing. Specifically, by interrogating and comparing up to 2688 parallel nucleic acid-protein interactions in vitro, we identified PNA triplex-bound factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We go on to suggest measures for improving PNA-mediated gene editing efficiencies and potential strategies for safe novel gene editing approaches. In this work, we also outline the development, characterization, and application of PNAs to modulate CRISPR-Cas9 activity and affinity for target genomic sequences. Based on rational antisense targeting of PNAs to guide RNA (gRNA) sequences, we describe methods to rapidly inhibit Cas9 for facile spatiotemporal control or to improve overall specificity and reduce deleterious off-target editing. This is a seminal study for the use of PNAs to modulate enzymatic activity and engineer nucleoproteins by direct antisense binding, and demonstrably improves gene editing safety and versatility without associated cellular toxicity. Finally, to understand the influence of novel metabolites on DNA repair deficiency in IDH mutant cancers, we use a computationally informed CRISPR-Cas9 design pipeline to correlate local methylation status with DSB repair at endogenous loci throughout the genome. Building on a body of prior work, we use this approach to establish the mechanism by which oncometabolites induce hypermethylation of histone 3 lysine 9 (H3K9) to mask local chromatin signaling required for proper DSB repair. This finding indicates potential strategies for targeted therapeutics in this prevalent class of cancers. Overall, this work establishes multiple advances for the improvement of gene editing tools and our ability to rationally manipulate DNA sequences in living cells. As part of a larger and continually evolving body of work, these projects progress our ability to develop and apply innovative, versatile, and safe next-generation gene editors for future human therapeutics and beyond.

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