ENGINEERED RECOMBINASES: TOOLS FOR THERAPEUTIC HUMAN GENOME EDITING.

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dc.contributor.advisorOwens, Jesse B.
dc.contributor.authorSato, Ryuei
dc.contributor.departmentDevelopmental & Reproductive Biology
dc.date.accessioned2024-07-02T23:42:14Z
dc.date.available2024-07-02T23:42:14Z
dc.date.issued2024
dc.description.degreePh.D.
dc.identifier.urihttps://hdl.handle.net/10125/108370
dc.subjectMolecular biology
dc.titleENGINEERED RECOMBINASES: TOOLS FOR THERAPEUTIC HUMAN GENOME EDITING.
dc.typeThesis
dcterms.abstractNumerous genetic human diseases will require precise and effective genome-targeting technologies to be treated in the future. Ideally, these technologies would be able to edit any genomic locus with high efficiency, high DNA sequence specificity, and few or no unintended side effects. Currently, a method for delivering a large DNA fragment to a single defined sequence in an efficient and safe manner does not exist. Current targeted approaches for gene editing that rely on CRISPR or other site-specific nucleases typically require double-strand DNA breaks (DSBs) that often generate unwanted byproducts or lead to chromosomal abnormalities. These technologies act passively; following the DNA break, rate-limiting host factors are needed to insert the donor DNA. Furthermore, because homologous directed repair (HDR) factors are only present during cell division, HDR is inefficient in non-dividing cells that make up most of the tissues of the body. Here, my studies will demonstrate effective methodologies to address these shortcomings in gene editing. I developed a system for actively inserting a large DNA sequence to a known target sequence. Rational mutations were incorporated into the endogenous DNA-binding domain of the piggyBac transposase to reduce non-specific binding and promote preferential binding and targeted insertion by catalytically dead Cas9 (dCas9). This strategy enabled us, for the first time, to direct transposition to the genome using RNA in human cells. Directed evolution could also improve proteins that are less characterized and are difficult to enhance using rational design. Using phage-assisted continuous evolution (PACE), we mutated the bacteriophage PhiC31 serine integrase to increase activity at the natural human pseudo site, Xq22.1 which meets the requirements to be considered a genetic safe harbor. Finally, we used PACE to increase the activity of two integrases, PhiC31 and Bxb1, on their native sites, resulting in mutant enzymes with a 10-fold increase in recombination. When combined with twin prime editing (PE), we were able to insert a 7kb donor into a single safe harbor loci in the human genome with efficiencies of more than 80% of available genomic targets. These technologies could be applicable to both preclinical research and potential gene replacement therapies for a multitude of genetic diseases.
dcterms.extent190 pages
dcterms.languageen
dcterms.publisherUniversity of Hawai'i at Manoa
dcterms.rightsAll UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
dcterms.typeText
local.identifier.alturihttp://dissertations.umi.com/hawii:12190

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Ph.D. - Developmental and Reproductive Biology