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Characterization of Pinpoint, a CRISPR/RNA-aptamer-mediated base editor, and its therapeutic potential in hemoglobinopathies
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Tan, Victor Ming Wei. Characterization of Pinpoint, a CRISPR/RNA-aptamer-mediated base editor, and its therapeutic potential in hemoglobinopathies. Retrieved from https://doi.org/doi:10.7282/t3-bh05-0n52

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TitleCharacterization of Pinpoint, a CRISPR/RNA-aptamer-mediated base editor, and its therapeutic potential in hemoglobinopathies
NameTan, Victor Ming Wei (author); Jin, Shengkan (chair); Ganesan, Shridar (member); Denzin, Lisa (member); Bunting, Samuel (member); Rutgers University; School of Graduate Studies
Date Created2023
Other Date2022-10 (degree)
SubjectPharmacology, Genetics, Base editing, CRISPR
Extent94 pages : illustrations
DescriptionGenome editing has traditionally relied on the formation of targeted DNA double strand breaks (DSB), followed by activation of DNA repair pathways such as homology-directed repair (HDR) and non-homologous end joining (NHEJ). Unfortunately, this approach is marred by (1) random insertions and deletions (indels) (NHEJ), (2) limited to proliferating cells and general lack of efficiency in non-dividing somatic cells (HDR). To overcome these limitations, we developed a base editing (BE) system that directly converts one type of DNA nucleotide bases into another. Our Pin-point BE strategy incorporates elements of Clusters of Regularly Interspaced Short Palindromic Repeats (CRISPR) and RNA-aptamer mediated recruitment. We engineered a nuclease-deficient Streptococcus pyogenes Cas9 nickase combined with RNA-aptamer containing sgRNA to recruit DNA/RNA editing enzymes capable of C·G→T·A conversions by cytidine deamination. We previously demonstrated effectiveness of our system on episomal DNA editing as well as endogenous gene editing on various cell models. To further improve on our system, we incorporated upgrades to our modular system in using various orthologous deaminases from other species. We then assessed their efficacies and editing capabilities on previously tested endogenous sites and characterized their Cas-dependent and Cas-independent off-target effects. To this end, we found several orthologs from Anolis carolinesis (green lizard) and Myotis lucifugus (bat) to have improved base editing efficiencies as well as differing editing window along the protospacer element.We further applied our BE technology for therapy against hemoglobinopathies such as beta-thalassemia and sickle cell disease (SCD). During human development, globin protein expression switches from fetal globin (?) to adult globin (β). Transcription proteins BCL11A and ZBTB7A bind ?-globin promoters at key stretches of DNA, repressing its expression upon maturation of the fetus to birth. Interestingly, naturally occurring mutations in these regions has been found in patients with milder symptoms. These mutations disrupt repressor binding to the ?-globin promoters, thus alleviating repression of the gene, leading to expression of fetal hemoglobin which compensates for deficient adult hemoglobin. We showed that our BE system is able to recapitulate these mutations, providing evidence of a promising therapy towards these hemoglobinopathies. Ultimately, we showed improvements to our BE technology as well as possible use for therapeutic applications, all independent of the DSBs and HDR necessary for previous technologies.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
Persistent URLhttps://doi.org/doi:10.7282/t3-bh05-0n52
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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