Project Summary: Genome editing tools provide methods to study and manipulate the genome. This has allowed for the development of cellular and animal-based models to study the pathological basis of many diseases. Over the last ten years, the rapid development of CRISPR-Cas based genome editing tools has driven advances in the biological sciences and has shown incredible utility in a wide range of fields ranging from basic research to clinical trials. Prime Editing (PE) is a newly developed CRISPR-Cas9 based tool that directly rewrites DNA sequences and has been shown to perform precise targeted insertions, deletions, and all 12 possible classes of point mutations without requiring DSBs. While this technology holds great promise, further study is needed to understand DNA repair mechanisms that result in productive editing and delivery strategies for the PE system to allow for broad applicability of this technology. In my preliminary studies, I have shown that modulation of key components of the PE system improves editing efficiencies. Additionally, I have condensed the published lentiviral system to a dual selection lentiviral system that is suitable for a wide range of cell types. The goal of this study is to further develop a PE platform capable of large fragment edits for functional sequence insertions. In aim 1, I will use structure-guided mutagenesis to generate a PE mutant better able to accommodate large fragment edits. In aim 2, I will use my generated tool to endogenously tag BRD4 isoforms with a functional sequence to characterize their roles in leukemia. Even though BRD4 is a therapeutic target with more than 20 inhibitors in clinical trials, these nonspecific drugs target several members of the BET family and all BRD4 isoforms, which are required for normal cellular function. Since this target lacks commercially available, isoform specific ChIP-grade antibodies and has unique targetable C terminal regions, it serves as a practical and disease relevant target to demonstrate proof of concept. Ultimately, development of this method will provide an addition to the CRISPR-Cas9 toolbox capable of numerous other tasks.