Rieffer, Amanda2025-01-282025-01-282023https://hdl.handle.net/11299/269606University of Minnesota Ph.D. dissertation. 2023. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Reuben Harris. 1 computer file (PDF); viii, 137 pages.Enzymes require specificity for substrates to overcome passive diffusion and efficiently conduct complex chemical reactions in biological systems. Only when binding to the correct substrate in the correct orientation will the reaction catalyze properly. Therefore, it is of utmost importance for researchers to determine the structural and biochemical relationships between their enzyme or substrate of interest to better understand how they function together to overcome the energy required to complete their respective chemical reactions. An example of this can be derived from nucleic acid binding proteins; for example, the APOBEC family of cytosine deaminases require specific ssDNA sequence motifs to efficiently bind to and conduct cytosine deamination reactions. In this thesis, we have developed a reporter system that can easily measure the efficiency of enzyme-catalyzed cytosine deamination in different dinucleotide contexts. This is done by tethering the cytosine deaminase to Cas9, a DNA-targeting enzyme, and directing it to a specific site in a reporter gene. Correction of a defective eGFP through a single cytosine-to-uracil deamination event results in the accumulation of fluorescence over time which can be measured via flow cytometry, fluorescence microscopy, and DNA sequencing. We compared several different members of the APOBEC family, both wild-type and engineered, and found that A3B has the best on-target dinucleotide specificity while A3A has high editing efficiency for each dinucleotide. Lastly, we optimized this assay to measure the proficiency of several APOBEC inhibitory technologies including viral proteins, small molecules, and substrate-based nucleic acids.enAPOBECbase editorCBEfluorescent assaygenome engineeringinhibitor screenThesis or Dissertation