Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. Here, we leveraged single-nucleus RNA-sequencing to generate a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type–specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified an immediate early gene expression program that is up-regulated following cocaine experience in vivo and dopamine receptor activation in vitro. Multiplexed induction of this gene program with a large-scale CRISPR-dCas9 activation strategy initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Together, these results define the transcriptional response to cocaine with cellular precision and demonstrate that drug-responsive gene programs can potentiate both physiological and behavioral adaptations to drugs of abuse.

Drug addiction is a worldwide health problem, with overdose rates of both psychostimulants and opioids currently on the rise in many developed countries. Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. However, even with well-studied drugs such as cocaine, druginduced transcriptional responses remain poorly understood due to the cellular heterogeneity of the NAc and complex drug actions via multiple neurotransmitter systems. Here, we leveraged highthroughput single-nucleus RNA-sequencing to create a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified specific neuronal subpopulations that are activated by cocaine, and defined an immediate early gene expression program that is upregulated following cocaine experience in vivo and dopamine (DA) receptor activation in vitro. To characterize the neuronal response to this DA-mediated gene expression signature, we engineered a large-scale CRISPR/dCas9 activation strategy to recreate this program. Multiplexed induction of this gene program initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and demonstrate that drug-responsive gene programs are sufficient to initiate both physiological and behavioral adaptations to drugs of abuse.NEARLY 5 MILLION Americans reported cocaine use in 2017, and recent increases in cocaine-related drug overdoses present significant public health challenges (1). A hallmark trait of drugs of abuse is the acute elevation of dopamine (DA) in the nucleus accumbens (NAc), a central integrator of the reward circuit (2-4). Abused drugs produce increases in DA that are greater in both concentration and duration than natural rewards (2,5,6), and this signaling is hypothesized to underlie maladaptive reinforcement after repeated drug use (7). Exposure to drugs of abuse results in significant transcriptional and epigenetic reorganization in the NAc (8-13), initiating synaptic and behavioral plasticity associated with the transition to drug addiction (7,14,15). However, even with well-studied drugs such as cocaine, drug-induced transcriptional responses remain poorly understood. This is in part due to the cellular heterogeneity of the NAc, which is a diverse structure containing multiple neuronal and non-neuronal subpopulations and complex neuronal circuitry. Additionally, many drugs of abuse engage multiple neurotransmitter systems in the NAc (16-22), and the specific contributions of DA-dependent transcriptional programs to neuronal physiology and behavior is not clear. Further, although drug experience leads to large scale transcriptional changes in the NAc, previou...

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective promoter human synapsin 1 enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.Correspondence to Jeremy Day (jjday@uab.edu | day-lab.org | @DayLabUAB)

Title (50-word maximum): A Cre-dependent CRISPR/dCas9 system for gene expression regulation in neuronsAbbreviated title (50-character maximum): Cre-dependent CRISPR system for neuronal gene regulation