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193276 https://hdl.handle.net/2286/R.2.N.193276 http://rightsstatements.org/vocab/InC/1.0/ http://creativecommons.org/licenses/by-nc-sa/4.0 2024-05 32 pages Kostrinsky, Jack Lapinaite, Audrone Mills, Jeremy Stephanopoulos, Nicholas Barrett, The Honors College Harrington Bioengineering Program Text Genome editing tools possess the potential to cure human genetic diseases. The most promising editing tools for addressing a significant portion of genetic diseases are adenine base editors (ABE). The complex consists of a deaminase domain fused to nCas9, guided by a sgRNA. Since no known adenine deaminase catalyzed DNA, E. coli tRNA deaminase (EcTadA) was evolved to ABE8e with 22 amino acid mutations to act on ssDNA substrates with extremely high efficiency. The cryoEM structure of ABE8e indicated that the evolved TadA is a dimer, and preliminary results show that wild-type TadA has weaker dimerization than TadA8e. This leaves us with the open question in the field: How does each amino acid introduced during directed evolution contribute to the dimerization strength and catalytic efficiency of ABE8e? In this experiment, we investigate the role of tyrosine 123 in dimerization strength as well as editing efficiency. With the reverse mutation of Y123 to H, the dimerization strength of TadA8e as well as the DNA editing efficiency of ABE8e were analyzed by ensemble FRET and in vitro single-turnover kinetics assays and were compared to ABE8e. The results from ensemble FRET reveal persistent dimer strength across varying protein concentrations indicating that Y123H does not impact the dimerization strength. The in vitro single-turnover kinetics assay revealed an editing efficiency and rate comparable to that of ABE8e which indicates that Y123H does not significantly impact the catalysis of ABE8e. Further experimentation, such as investigation of mutations that are close in proximity or introduced simultaneously during directed evolution, is required to understand the role of tyrosine 123 in ABE8e. base editor gene editing Molecular Biology base editor cas9 Understanding the Molecular Mechanism of DNA Adenine Base Editor: Functional Role of Tyrosine 123 in the Deaminase Domain