Optimization of CRISPR/Cas-based Programmable DNA Methylation

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dc.contributor.advisorHilton, Isaacen_US
dc.contributor.advisorBashor, Caleben_US
dc.creatorGuerra Resendez, Rosa Seleniaen_US
dc.date.accessioned2024-05-21T21:52:17Zen_US
dc.date.created2024-05en_US
dc.date.issued2024-04-05en_US
dc.date.submittedMay 2024en_US
dc.date.updated2024-05-21T21:52:17Zen_US
dc.descriptionEMBARGO NOTE: This item is embargoed until 2026-05-01en_US
dc.description.abstractCRISPR/Cas-based epigenome editing systems have transformed biomedical research by providing transcriptional and epigenetic control of healthy and disease states. Even though synthetic biology tools for DNA methylation writing and erasing have been employed in different architectural configurations and recruitment strategies, efforts to improve their functionality have been limited. Thus, engineering approaches are underexplored and needed to enhance the enzymatic activity of these tools, in particular for therapeutic applications in primary cells and in vivo models. In this doctoral dissertation, I present the optimization and application of programmable DNA methylation in human cells using CRISPR/Cas9-based epigenome editing. In the first chapter, optimization of lentiviral delivery of CRISPR/Cas9-based epigenome editing tools in human cells was conducted to better understand the parameters that lead to efficient delivery of epigenome editing tools. In the second chapter, rational engineering of the programmable synthetic DNA methylation writer (dCas9-DNMT3A/3L) was used to characterize the differential DNA methyltransferase activity of each designed variant, which catalyzed a distinctive methylation deposition profile and magnitude which correlated to gene silencing in different human cell lines. In the third chapter, engineered variants of dCas9-DNMT3A/3L were delivered to human primary T cells in an attempt to model immune exhaustion by targeting the BATF3 gene. Transcriptional silencing of BATF3 led to reduced cytokine production in human primary T cells. However, further experiments are needed to confirm that the reduced gene expression is caused by the DNA methylation deposited by the dCas9-based epigenetic effectors or if other factors independent of this epigenetic mark might be affecting transcription at this locus. Overall, the use of rationally engineered variants of the dCas9-DNMT3A/3L present an opportunity to interrogate the mechanistic parameters involved in programmable DNA methylation deposition and enable tunable transcriptional repression in human cells.en_US
dc.embargo.lift2026-05-01en_US
dc.embargo.terms2026-05-01en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationResendez, Rosa Guerra. Optimization of CRISPR/Cas-based Programmable DNA Methylation. (2024). PhD diss., Rice University. https://hdl.handle.net/1911/116140en_US
dc.identifier.urihttps://hdl.handle.net/1911/116140en_US
dc.language.isoengen_US
dc.rightsCopyright 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.en_US
dc.subjectsynthetic biologyen_US
dc.subjectepigenome editingen_US
dc.subjectguide RNAen_US
dc.subjectCRISPRen_US
dc.subjectdeactivated Cas9en_US
dc.subjectDNA methylationen_US
dc.titleOptimization of CRISPR/Cas-based Programmable DNA Methylationen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentSystems, Synthetic and Physical Biologyen_US
thesis.degree.disciplineSystems/Synthetic/Phys Biologyen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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