All ETDs from UAB

Advisory Committee Chair

Jeremy J Day

Advisory Committee Members

Robert Kesterson

Aurelio Galli

Linda Overstreet-Wadiche

Trygve Tollefsbol

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Drugs of abuse increase dopamine concentrations in the nucleus accumbens, a key reward structure that integrates contextual and cue-related information and regulates motivated behavior. This surge of dopamine triggers cell signaling cascades that converge in the nucleus to cause changes in gene expression, which are thought to lead to the observed functional and structural alterations in the reward circuit after exposure to drugs of abuse. However, while various drugs of abuse cause transcriptional changes, previously available tools have not had the capacity to deeply characterize these gene programs. Therefore, we optimized a dual lentivirus CRISPR system for targeted gene modulation via fusion of a transcriptional activator (VPR) (CRISPRa) to a catalytically dead Cas9 (dCas9). We found this system effectively drives expression at many gene targets and provides titratable gene expression. Additionally, CRISPRa is capable of simultaneously targeting multiple genes at once and robustly increases protein expression of a target gene in vivo. Here, we defined a dopamine-regulated gene expression program in cultured striatal neurons and engineered a large-scale multiplexed CRISPR activation strategy to recreate this program. Induction of dopamine-responsive genes generated a secondary synapse-centric transcriptional wave and altered striatal physiological properties in vitro, and enhanced cocaine sensitization in vivo. These results provide proof of principle evidence that activity-dependent gene programs are sufficient to initiation both physiological and behavioral adaptations. Broadly, these findings indicate that this neuron-optimized CRISPR-based transcriptional modulation system will enable specific and large-scale control of gene expression profiles within the CNS to elucidate the role of gene expression in neuronal function, behavior, and neuropsychiatric disorders.

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