Epigenome Editing in Opioid Action

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Project Summary The goal of this project is to develop CRISPR epigenome editing tools that achieve cell type specificity and in some cases temporal specificity to target opioid regulated molecules. Opioid use, dependence, and addiction have dramatically increased to epidemic proportions in recent years, leading to substantial financial and societal health burdens, as well as an increasing number of overdoses. The number of encounters with synthetic opioids, primarily fentanyl, has increased 300% since 2014, resulting in an increase of 72% in synthetic opioid overdoses. To combat this epidemic it is imperative that we understand the neurobiological underpinnings that lead to opioid use disorder. We must identify disrupted neuron subtypes in the brain in opioid use disorders and dysregulated molecules within these neurons that underlie cellular, circuit, and ultimately behavioral adaptations. We must then develop more effective tools to target endogenous gene transcription of molecules disrupted in opioid used disorder. To do this we performed RNA-seq in nucleus accumbens medium spiny neuron subtypes, those expressing D1 or D2, in fentanyl abstinent animals, during a time point in which they display stress vulnerable behavior. We identified key gene expression networks that are altered in MSN subtypes of fentanyl abstinent mice. In this proposal we will develop CRISPR epigenome editing tools, including temporal specific Optogenetic CRISPR tools, for selective cell subtype targeting of key fentanyl regulated genes in D1- MSNs and D2-MSNs. We will test these tools in Neuro2a cells and then in vivo in MSN subtypes (R21). Validated tools will be used in vivo to alter major hub genes, highly connected genes that regulate expression of other genes within a network. RNA-seq on MSN subtypes during fentanyl abstinence will determine if CRISPR tools are effective in targeting hub genes to alter their gene co- expression networks (R33). In parallel we will test Opto-CRISPR tools to disrupt hub genes during discreet stress susceptible and opioid intake and relapse time points (R33). Collectively, our studies have the ability to uncover unique opioid molecular mechanisms by targeting the epigenome at endogenous gene loci. Importantly our developed tools can be applied across all brain regions, cell subtypes, and neuroscience disciplines.