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195366 https://hdl.handle.net/2286/R.2.N.195366 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2024 2026-08-01T16:05:32 146 pages Doctoral Dissertation Academic theses Text eng CHEN, XIAOYU Lapinaite, Audrone AL Levitus, Marcia ML Stephanopoulos, Nicholas NS Arizona State University Partial requirement for: Ph.D., Arizona State University, 2024 Field of study: Biochemistry Clustered regularly interspaced short palindromic repeats (CRISPR)-based DNA Adenine Base Editors (ABEs) represent a groundbreaking advancement in precision genome editing, holding great potential for treating human genetic diseases caused by single nucleotide polymorphisms (SNPs). ABE8e, the most efficient ABE to date, catalyzes the conversion of adenine to guanine, introducing targeted point mutations into genomic DNA. This technology leverages the fusion of CRISPR-Cas9 molecular machinery with a single-stranded DNA (ssDNA)-specific adenosine deaminase evolved from a bacterial tRNA-specific adenosine deaminase (TadA) through directed evolution. This interdisciplinary study, employing computational (molecular dynamics simulations and free energy simulations) and experimental [ensemble Förster Resonance Energy Transfer (FRET), thermal stability assessments, and in vitro activity assays] approaches, reveals the biophysical foundations underlying ABE8e’s 500-fold increase in DNA base editing efficiency compared to other generations. Here the key to this enhancement is shown to be the stable dimerization of the deaminase domain (TadA8e). Its strategic juxtaposition to Streptococcus pyogenes Cas9 (SpCas9) and DNA substrate is achieved via critical interactions involving residues in the TadA8e docking domain (R98 and R129), the residues in the RuvC domain of SpCas9 (E1046) and the phosphates in the backbone of the DNA substrate, uniquely established when TadA8e operates as a stable dimer. It is revealed that T111R and D119N in combination with N122H, mutations introduced to TadA during the evolution of ABE7.10 to ABE8e, drive TadA8e dimerization and enhance DNA editing efficiency of ABE8e. However, ABE8e’s dimerization and aggregation hinder its delivery to the cell using cell-penetrating peptides (CPPs). Thus, a monomeric form of ABE8e is engineered by disrupting the hydrophobic dimerization interface of TadA8e using the QTY code, however, the TadA8e dimers persisted. Ongoing computational simulations aim to identify additional critical residues for efficient disruption of TadA8e dimer.Overall, these findings illuminate the molecular mechanisms driving ABE8e’s improved performance and suggest new engineering strategies aimed at mitigating off-target effects, enhancing editing efficiency, and streamlining cell delivery processes all of which are crucial for the therapeutic application of precision genome editors. Biochemistry Understanding Molecular Mechanisms of DNA Adenine Base Editors to Advance Precision Gene Therapies