Chromatin structure, accessibility, and the spatial organization of DNA within the nucleus play essential roles in gene regulation and contribute to cellular heterogeneity. There is also growing interest in understanding how genome editing affects these features. Developing new genomic tools can help elucidate this complex system, and DNA-modifying enzymes offer powerful biochemical strategies by introducing sequence changes that can be captured through sequencing. In this thesis, I develop and apply novel chemical genomic tools, primarily using double-stranded DNA cytidine deaminases, to advance our understanding of chromatin structure and gene regulation. Chapter 1 provides relevant backgrounds including the advantages and limitations of existing methods. In Chapter 2, I present a new approach that we developed to measure chromatin accessibility: Targeted Deaminase-Accessible Chromatin sequencing (TDAC-seq). Cis-regulatory elements regulate gene expression, and CRISPR-based genome editing of these regions has enabled breakthrough gene therapies for diseases such as sickle cell disease. Understanding how genome editing affects chromatin accessibility is crucial for elucidating underlying molecular mechanisms. However, existing technologies that simultaneously perturb and map cis-regulatory elements have significant limitations in detecting endogenous edits and linking them to chromatin accessibility with high resolution and throughput. TDACseq addresses this by using variants of dsDNA cytidine deaminase (DddA) to mark accessible regions through cytidine deamination. These edits are detected as C•G-to-T•A mutations after targeted long-range PCR and long-read sequencing. TDAC-seq thereby provides single-molecule, single-nucleotide resolution of chromatin accessibility and protein footprints across target regions. Furthermore, TDAC-seq simultaneously captures chromatin accessibility and CRISPR edits from the same DNA molecules, and we combined TDAC-seq with pooled CRISPR-Cas9 and adenosine base editor screens targeting the HS2 enhancer of the β-globin locus. We further expanded this method to perform a large-scale pooled CRISPR screen targeting an enhancer downstream of GFI1B linked to myeloproliferative neoplasm risk in CD34+ hematopoietic stem and progenitor cells, identifying key regulatory motifs of this enhancer. These results iv highlight the scalability and resolution of TDAC-seq for fine-mapping sequence-function relationships of target cis-regulatory elements. Chapter 3 discusses the broader utility of DddA-based mutagenesis. Because DddA-induced edits are PCR-compatible, they can be easily combined with other sequencing strategies. I describe additional applications that we have explored, as well as those reported by other research groups. These applications include recruiting DddA11 to single-stranded DNA regions via N3-kethoxal labeling to mark transcription bubbles, site-specific labeling of chromatin-associated factors using DddA fusion proteins, and integrating DddA mutagenesis with single-cell sequencing to reveal transcription factor footprints. I also propose several future directions for expanding the chemical genomic toolkit using dsDNA deaminases, emphasizing their versatility for probing genome structure and regulation. Appendix A describes Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C), another method we developed that jointly measures chromosome conformation, DNA methylation, and lamina positioning by fusing the GpC cytosine methyltransferase M.CviPI to Lamin B1. This GpC methylation signature was integrated into a bisulfite Hi-C workflow to study the role of H3K27me3 at the boundaries of laminaassociated domains and compartments. Overall, this thesis demonstrates multiple approaches for using DNA-modifying enzymes to develop novel chemical genomic tools for studying genome structure. These new tools generate unique datasets unachievable through existing methods and provide new insights that advance our understanding of gene regulatory mechanisms.