A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

Savell, Katherine E.; Bach, Svitlana V.; Zipperly, Morgan E.; Revanna, Jasmin S.; Goska, Nicholas A.; Tuscher, Jennifer J.; Duke, Corey G.; Sultan, Faraz; Burke, Julia N.; Williams, Derek M.; Ianov, Lara; Day, Jeremy J.

doi:10.1523/eneuro.0495-18.2019

Cited by 107 publications

(128 citation statements)

References 65 publications

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“…4i-j, Supplemental Figure 9), a region critical for the development and maintenance of sensitization (48,49). We previously validated robust and neuron-selective expression of this CRISPRa system in vivo within 2 weeks of viral delivery into the NAc, and demonstrated that this tool results in elevated protein levels of target genes (35). Eighteen days after viral infusion, rats underwent locomotor sensitization testing in a novel environment using a dose of cocaine (10mg/kg) that does not reliably produce robust sensitization (50,51).…”

Section: Controlmentioning

confidence: 94%

“…However, drugs of abuse target many distinct receptor classes in a variety of neuronal and non-neuronal cell types (20,21), and these complex drug actions make identification of DA-induced gene expression programs difficult using in vivo models. Therefore, we took advantage of a well-studied and controllable rat primary striatal neuron culture system (35,36) to enable comprehensive and specific identification of DA-dependent gene expression programs in MSNs. Using this system, we first identified a core signature of the transcriptional response to DA receptor activation by performing deep RNA-seq on bulk striatal neuronal cultures treated with 1µM DA for 1hr, a treatment that closely models concentrations and temporal duration of DA increases found in vivo after acute cocaine exposure (2,3,6).…”

Section: Genesmentioning

confidence: 99%

“…Egr3 Egr2 log2(Fold change) Target/LacZ log2(Fold change) Dopaplex/LacZ enabled rapid and multiplexable transcriptional control in the mammalian central nervous system, providing an avenue to investigate how gene programs regulate normal and maladaptive brain states (35,(42)(43)(44). To simultaneously activate multiple genes in this DA-induced gene expression profile, we harnessed a neuron-optimized CRISPR activation (CRISPRa) system ( Fig.…”

Section: Controlmentioning

confidence: 99%

“…To simultaneously activate multiple genes in this DA-induced gene expression profile, we harnessed a neuron-optimized CRISPR activation (CRISPRa) system ( Fig. 3) activator VPR (a concatemer of the herpes simplex viral protein VP16, the p65 subunit of NF-kB, and the gammaherpesvirus transactivator Rta) (35,45). This system allows multiplexed gene programming through the design of single guide RNAs (sgRNAs) that target the promoter region of selected genes.…”

Section: Controlmentioning

confidence: 99%

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A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Savell

Zipperly

Tuscher

et al. 2019

Preprint

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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...

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Section: Controlmentioning

confidence: 94%

Section: Genesmentioning

confidence: 99%

Section: Controlmentioning

confidence: 99%

Section: Controlmentioning

confidence: 99%

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A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Savell

Zipperly

Tuscher

et al. 2019

Preprint

Self Cite

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“…Innovations and adaptations of CRISPR/Cas9 technologies comprise the inclusion of LoxP/Cre sites on recombination templates (to allow removal of selection markers from established knock-in/out lines) and the SapTrap system derived from GoldenGate cloning, which provides a simple way to make a variety of knock-in/out repair constructs (Au et al, 2019;Schwartz & Jorgensen, 2016). CRISPR interference and CRISPR activation (CRISPRi and CRISPRa, respectively) have also been successful in C. elegans (Long et al, 2015;Savell et al, 2019).…”

Section: Of 35mentioning

confidence: 99%

Working with Worms: Caenorhabditis elegans as a Model Organism

Meneely

Dahlberg

Rose

2019

CP Essential Lab Tech

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Since its introduction as a laboratory organism 50 years ago, the nematode worm Caenorhabditis elegans has become one of the most widely used and versatile models for nearly all aspects of biological and genomic research. Many experiments in C. elegans begin with the generation and analysis of mutants that affect a specific biological process, so genetic techniques are the foundation of worm research. Many different aspects of biology are being studied in C. elegans, and three different recent Nobel Prizes have recognized six researchers working with worms. In addition, C. elegans was the first multicellular organism to have its genome sequenced, so many of the standard genomic methods have also been pioneered in C. elegans. In fact, many novel techniques and ideas are initially tested in C. elegans because of its versatility as a research organism. It is also appropriate for introducing undergraduate students to research, and some of its strengths and challenges for this purpose are discussed. © 2019 The Authors.

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Epigenetic editing: Dissecting chromatin function in context

Policarpi

Dabin

2021

BioEssays

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How epigenetic mechanisms regulate genome output and response to stimuli is a fundamental question in development and disease. Past decades have made tremendous progress in deciphering the regulatory relationships involved by correlating aggregated (epi)genomics profiles with global perturbations. However, the recent development of epigenetic editing technologies now enables researchers to move beyond inferred conclusions, towards explicit causal reasoning, through 'programing' precise chromatin perturbations in single cells. Here, we first discuss the major unresolved questions in the epigenetics field that can be addressed by programable epigenome editing, including the context-dependent function and memory of chromatin states.We then describe the epigenetic editing toolkit focusing on CRISPR-based technologies, and highlight its achievements, drawbacks and promise. Finally, we consider the potential future application of epigenetic editing to the study and treatment of specific disease conditions.

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A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

Cited by 107 publications

References 65 publications

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Working with Worms: Caenorhabditis elegans as a Model Organism

Epigenetic editing: Dissecting chromatin function in context

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