Electronic Theses and Dissertations

Date

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biomedical Engineering

Committee Chair

Gary L Bowlin

Committee Member

Alexander C Espinosa

Committee Member

Richard Smith

Committee Member

Bernie Daigle

Abstract

Spatiotemporal organization and regulation of genomic and epigenomic processes is a phenomenon central to life. The pursuit of knowledge about these processes often requires specialized tools to be able to dissect biological mechanisms. CRISPR/Cas has emerged as a powerful tool for genome engineering, and has seen widespread use. Coupling CRISPR/Cas systems with tools that allow for spatiotemporal control is predicted to be transformational to the ability to perturb systems and gain the insights necessary to understand a diverse set of biological questions, and ultimately treat some of the most pervasive and elusive diseases. This dissertation describes the development of an optogenetic system to harness the full potential of CRISPR/Cas systems. To this end, we present BluVIPR, a system to control guide RNA production for precise spatiotemporal control of orthogonal CRISPR/Cas systems. We make special emphasis on the versatility of the system, and the compatibility with current mouse models that are stalwarts of biomedical research. We then show a proof of concept study for the delivery of light gradients to cells engineered with the BluVIPR system via air gap electrospun templates, envisioning how biomaterial-guided optogenetics could be used as a therapeutic strategy for interfacial tissue engineering. We finally show how the versatility and orthogonality of BluVIPR would allow for the development of a synthetic gene regulatory network, composed of a simple, genetically-encoded, digital demultiplexer circuit, to interpret light gradients as a cue for opposing gradient production of growth factors. We believe that this optogenetic CRISPR/Cas system is an important contribution to the toolkit of diverse biological fields of research, and envision many exciting applications in tissue engineering, tumor immunology, developmental biology, and beyond.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to ProQuest

Notes

Open Access

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