Gao, XueVerduzco, Rafael2024-05-222024-052024-04-17May 2024Zeng, Hongzhi. Advancing Precision and Controllable Molecular Tools for Genetic Engineering and Disease Treatment. (2024). PhD diss., Rice University. https://hdl.handle.net/1911/116193https://hdl.handle.net/1911/116193EMBARGO NOTE: This item is embargoed until 2026-05-01The emergence of programmable gene editing tools has transformed life sciences by empowering researchers to execute precise and targeted genomic alterations in living cells. The advent of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology has greatly accelerated genome editing research and applications. However, CRISPR-Cas faces limitations due to the low efficiency of homology-directed repair after Cas nuclease- induced double-stranded DNA breaks (DSBs), which can result in unintended genomic alterations and raise safety concerns. Base editors (BEs), leveraging a catalytically impaired nuclease and a single-stranded DNA deaminase enzyme, offer a promising alternative by facilitating targeted point mutations without requiring DSBs or donor DNA templates. However, the challenge of off-target effects and the lack of temporal control over BE activity when delivered via viral vectors remain significant hurdles. My thesis describes two projects that I lead: (1) A split and inducible adenine base editor for precise in vivo base editing, and (2) Precision A3G base editors and prime editors for cystic fibrosis modeling and correction. These projects explore the underlying principles of BEs, detailing engineering strategies for achieving small-molecule-controlled base editing in vivo and outline efforts to enhance the specificity of adenine and cytosine base editors. Additionally, these projects involve viral and non-viral delivery methods for BEs, emphasizing their applications in human disease modeling, treatment, and prevention. Lastly, my projects contain applications of prime editing technology, a more versatile gene-editing tool than BE, highlighting its promise for biological research and therapeutic applications.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.CRISPRBase EditingGene TherapyProtein EngineeringGenetic EngineeringThesis2024-05-22