Optimization of CRISPR/Cas-based Programmable DNA Methylation

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dc.contributor.advisorHilton, Isaac
dc.contributor.advisorBashor, Caleb
dc.creatorGuerra Resendez, Rosa Selenia
dc.date.accessioned2024-05-21T21:52:17Z
dc.date.created2024-05
dc.date.issued2024-04-05
dc.date.submittedMay 2024
dc.date.updated2024-05-21T21:52:17Z
dc.descriptionEMBARGO NOTE: This item is embargoed until 2026-05-01
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.
dc.embargo.lift2026-05-01
dc.embargo.terms2026-05-01
dc.format.mimetypeapplication/pdf
dc.identifier.citationResendez, Rosa Guerra. Optimization of CRISPR/Cas-based Programmable DNA Methylation. (2024). PhD diss., Rice University. https://hdl.handle.net/1911/116140
dc.identifier.urihttps://hdl.handle.net/1911/116140
dc.language.isoeng
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.
dc.subjectsynthetic biology
dc.subjectepigenome editing
dc.subjectguide RNA
dc.subjectCRISPR
dc.subjectdeactivated Cas9
dc.subjectDNA methylation
dc.titleOptimization of CRISPR/Cas-based Programmable DNA Methylation
dc.typeThesis
dc.type.materialText
thesis.degree.departmentSystems, Synthetic and Physical Biology
thesis.degree.disciplineSystems/Synthetic/Phys Biology
thesis.degree.grantorRice University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
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