Thesis advisor: Eranthie Weerapana / Reactive oxygen species (ROS) modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways especially within ROS generating organelles such as the mitochondria. There are several known examples of mitochondrial cysteine targets that alter protein and pathway activity resulting in pathological effects. Several chemoproteomic workflows, including ABPP and OxICAT, can be used to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular regions and ROS hotspots remains challenging with existing workflows. Here, we present combined cysteine- monitoring chemoproteomic platforms (isoTOP-ABPP and OxICAT) with mitochondrial enrichment (organelle isolation and proximity labeling) to monitor cysteine oxidation events within the mitochondria. First, we profile redox- sensitive cysteines under exogenous and endogenous peroxide in isolated mitochondria using isoTOP-ABPP and OxICAT. Next, we introduce PL-OxICAT which combines enzymatic proximity labeling (PL) (TurboID/APEX) and OxICAT to monitor localized cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space as well as ROS hotspots. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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A bacterial and archaeal adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), has recently been engineered for genome editing. This RNA-guided platform has simplified genetic manipulation and holds promise for therapeutic applications. However, off-target editing has been one of the major concerns of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Despite extensive enzyme engineering to reduce off-target editing of SpyCas9, we have turned to nature and uncovered a Cas9 ortholog from Neisseria meningitidis (Nme) with high fidelity. In the first part of my thesis, we have systematically characterized Nme1Cas9 for engineering mammalian genomes and demonstrated its high specificity by genome-wide off-targeting detection methods in vitro and in cellulo, and thus provided a new platform for accurate genome editing. Due to its flexibility, CRISPR is becoming a versatile tool not only for genome editing, but also for chromatin manipulation. These alternative applications are possible because of the programmable targeting capacity of catalytically dead Cas9 (dCas9). In the second part of my thesis, we have combined dCas9 with the engineered plant enzyme ascorbate peroxidase (APEX2) to develop a proteomic method called dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST). Relying on the spatially restricted, fast biotin labeling of proteins near defined genomic loci, C-BERST enables the high-throughput identification of known telomere- and centromere- associated proteomes and novel factors. Furthermore, we have extended C-BERST to map the c-fos promoter and gained new insights regarding the dynamic transcriptional regulation process. Taken together, C-BERST can advance our understanding of chromatin regulators and their roles in nuclear and chromosome biology.