OPTOGENETIC GUIDE RNA PRODUCTION FOR SPATIOTEMPORAL REGULATION OF CRISPR/CAS SYSTEM

Velasquez Pulgarin, Diego Augusto

English

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

University of Memphis Digital Commons

OPTOGENETIC GUIDE RNA PRODUCTION FOR SPATIOTEMPORAL REGULATION OF CRISPR/CAS SYSTEMS

https://digitalcommons.memphis.edu/cgi/viewcontent.cgi?article=3931&context=etd

OPTOGENETIC GUIDE RNA PRODUCTION FOR SPATIOTEMPORAL REGULATION OF CRISPR/CAS SYSTEMS

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University of Memphis Digital Commons