Neuroepigenetic Editing

Abstract

Epigenetic regulation is intrinsic to basic neurobiological function as well as neurological disease. Regulation of chromatin-modifying enzymes in the brain is critical during both development and adulthood and in response to external stimuli. Biochemical studies are complemented by numerous next-generation sequencing (NGS) studies that quantify global changes in gene expression, chromatin accessibility, histone and DNA modifications in neurons and glial cells. Neuroepigenetic editing tools are essential to distinguish between the mere presence and functional relevance of histone and DNA modifications to gene transcription in the brain and animal behavior. This review discusses current advances in neuroepigenetic editing, highlighting methodological considerations pertinent to neuroscience, such as delivery methods and the spatiotemporal specificity of editing and it demonstrates the enormous potential of epigenetic editing for basic neurobiological research and therapeutic application.

1. Introduction

Regulation of gene expression is a fundamental mechanism of nervous system development and plasticity. Like other cells, neurons possess a genome capable of expressing programmed output. However, neurons are unique in the degree to which they respond and adapt to external stimuli. Epigenetic regulation of neuronal gene expression is a fundamental mechanism by which environmental stimuli are transmitted into neurobiological substrates, capable of driving behavior in both health and disease. This review focuses on the emerging field of neuroepigenetic editing, in which specific DNA or histone modifications are targeted to a single genomic locus. As such, neuroepigenetic editing is an approach to causally link such modifications to altered neuronal gene expression, physiology, and animal behavior.

Epigenetic mechanisms of gene regulation include the expression and activity of histone-modifying enzymes, global regulation of histone posttranslational modifications (hPTMs), and DNA methylation (DNAme), as well as regulation of noncoding small and long RNAs [1–3]. These mechanisms elicit defined programs of gene expression during development and throughout life, as well as respond to sensory experience. Stable biochemical modifications to chromatin and DNA elicit long-term changes in gene expression that underlie various forms of plasticity and memory. The nervous system responds to environmental stimuli throughout life, and the integration of such signals is intrinsic to healthy development and behavior. Environmental stimuli can alter the neuronal epigenome, can be persistent, and regulate transcriptional responses to future stimuli. As such, epigenetic mechanisms underlie the transcriptional memory necessary for the nervous system to develop and adapt.

Expression of neuronal chromatin and DNA modifying enzymes is regulated across development and adulthood, both basally and in response to external stimuli [4–6]. Classic biochemistry and next-generation sequencing (NGS) studies are used to quantify hPTMs and open chromatin in neurons and other cell types in the nervous system. Genome-wide analyses of individual cells and nuclei have emerged in the last decade to elucidate the epigenetic landscapes of individual neuronal and nonneuronal subpopulations. The epigenome is regulated across mammalian development [7–10], learning and memory [11–13], and neurological and psychiatric disease [14–16].

While NGS and biochemical studies strongly implicate epigenetic reprogramming in neuronal gene expression, experience-dependent deposition of hPTMs at a gene of interest is analyzed in the context of global chromatin changes. Thus, it is not possible to discern the primary action of a given epigenetic modifier at a specific gene from indirect, downstream regulation by pleiotropic activity across the genome. Epigenetic editing is a strategy in which a given epigenetic modification is targeted to a single genomic locus [17–19]. As such, epigenetic editing is a primary approach to discriminate between the mere presence and causal relevance of histone and DNA modifications to gene expression. Furthermore, such tools can be used to identify new gene regulatory elements, such as distal promoters and enhancers [20, 21], the genetic diversity of which has been linked to aberrant neurobiological function [7, 22–26]. This review summarizes studies that apply locus-specific epigenetic editing to the nervous system.

Several unique features of the nervous system underscore the utility of neuroepigenetic editing to elucidate mechanisms of gene regulation. First, prior studies have largely relied on exogenous overexpression of a gene of interest, delivered either virally or genetically. Both methods drive expression of the gene of interest to a much greater extent than the physiological range of gene activation, which is typically between one- and twofold from basal levels for most regulated genes in post-mitotic neurons. By reprogramming endogenous mechanisms of gene activation, neuroepigenetic editing has the capacity to drive biologically relevant gene expression. Second, neurobiological application typically requires the targeting of endogenous genes rather than reporter systems, promoting (1) the elucidation of bona fide mechanisms of experience-dependent gene regulation and (2) the potential therapeutic applications of neuroepigenome engineering. Finally, epigenetic editing lends itself to studies of chronic changes in gene expression, as these tools can potentially deposit long-lasting modifications to histones and DNA. This approach can therefore be used to examine persistent mechanisms of gene regulation that underlie neurobiological function.

2. Epigenetic Editing Tools

There exist several excellent reviews of the various methods of epigenetic editing [19, 27, 28], which cover tools that target reporter systems as well as endogenous loci. This review will focus specifically on epigenetic editing in neurons, particularly applied to endogenous genes. The majority of the published literature utilizes gene expression profiling to assess the efficacy of neuroepigenetic editing tools, but a few assess chromatin modifications as well [26, 29–32]. These latter examples allow conclusions to be drawn on the causal transcriptional relevance of specific histone modifications. Fig. 1 illustrates the main tools available for epigenetic editing.

Fig. 1.

Epigenetic editing tools. Three main approaches to epigenetic editing have been applied in neurons. ZFPs and TALEs rely on a protein DNA-binding domain tethered to an effector domain, while the CRISPR/dCas9 system utilizes an RNA–DNA interaction to guide the dCas9-effector domain fusion to the target gene. Effector domains act to modify histone tails and/or DNA at target loci. ZFPs are shown with Zn²⁺ stabilizing ion (grey dot) and TALEs are shown with C- and N-terminal domains in yellow

The first study of epigenetic editing at an endogenous locus, exemplifying many of the themes of this review, utilized zinc-finger proteins (ZFPs) targeting the human vascular endothelial growth factor (VEGF) gene in cultured human embryonic kidney (HEK) 293T cells [33]. ZFPs designed to recognize and bind the VEGF promoter were fused to a truncated version of the histone methyltransferase, G9a, to repress VEGFA expression via deposition of H3 lysine 9 dimethylation (H3K9me2), the mark catalyzed by G9a. ZFP-G9a activity, as well as that of a catalytic mutant version, was assayed by in vitro methylation of purified histone H3 and quantitative chromatin immunoprecipitation (qChIP) of cellular histones, establishing the utility of this functional domain in epigenetic editing. Importantly, while ZFP-G9a effectively repressed expression of endogenous VEGFA in cell culture, it was not effective in repressing expression from a luciferase reporter plasmid, indicating that the function of this repressor relies on a chromatinized genomic context. Because methylation of H3K9 leads to a spreading of repressive methylation via recruitment of HP1, the authors analyzed H3K9me2 at sites 500 bp up- and downstream of the ZFP binding site and found that, indeed, ZFP-G9a induced H3K9me2 enrichment at sites both proximal and distal to the ZFP binding site [33]. This result was the first to establish that targeting an exogenous histone methyltransferase to an individual gene can regulate the endogenous transcriptional machinery.

Transcription activator-like effectors (TALEs) are similar to ZFPs in that they rely on a protein–DNA interaction, determined by the primary amino acid structure [34]. TALEs, derived from plant pathogenic bacteria, consist of a DNA-binding domain that contains tandem repeats of 34-aa sequences (termed monomers) that are required for DNA recognition and binding. Each TALE DNA-binding monomer contains a tandem repeat variable domain (RVD) which can bind to one base pair in the DNA [35]. Several groups have published protocols and resource papers on TALE and ZFP design and cloning, as well as open-source platforms for in-silico binding assays [34, 36–38].

The most recently developed and widely applied tool for neuroepigenetic editing is derived from the prokaryotic RNA-guided endonuclease, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system [39–42]. Cas9 can be targeted to a specific genomic locus of interest using a rationally designed RNA guide, or single guide RNA (sgRNA). A nuclease-deficient Cas9 (dCas9) fused to an effector domain can thus be used as an epigenetic editing tool. The ease of design and synthesis as well as the scalability of the CRISPR-Cas9 system has led to its use for locus-specific neuroepigenetic editing in many studies, as reviewed below.

With regard to the selection of editing tools, the specificity of target sequence binding is perhaps the first consideration. In this respect, accessibility of the target site is of particular importance with respect to epigenetic editing of chromatinized endogenous loci in neurons. For example, restriction enzyme or DNAse1 hypersensitivity mapping of “open chromatin” can be an efficient means to select accessible target sites. One study utilized this approach to design ZFP-Kruppel-associated box (KRAB) and ZFP-p65 proteins to repress and activate the CDK2 gene, respectively, and demonstrated target gene-specific repression using a microarray [43]. With respect to target specificity, a small number of publications report comparisons of the various tools within a single study [31, 44, 45]. Genome-wide mapping of ZFP, TALE, and CRISPR/Cas9 binding by ChIP-sequencing (ChIP-seq) has revealed substantial off-target localization of DNA binding domains [45–49]. However, because these proteins rely on genome scanning to find their putative binding sites, ChIP-seq leads to false positive identification. That is, off-target localization rarely corresponds to changes in gene transcription or chromatin accessibility [47, 48, 50, 51]. For example, ChIP-seq analysis of affinity-tagged, NFD (no functional domain)-ZFPs found ~25,000 off-target binding sites, yet less than 2.8% of these target sites correlated with changes in nearby gene expression by RNA-seq [48]. Additional studies of specificity found that increased off-target zinc-finger nuclease (ZFN) activity is correlated with the concentration of transfected ZFN [52], while Cas9 off-target interactions can be computationally predicted based on frequency of highly similar genomic target sites [53]. Furthermore, considering the evidence that these tools function within specific epigenetic contexts [33], off-target effects would require improbable off-target DNA binding to gene regions of similar epigenetic microenvironments to the targeted, high-affinity site (i.e., gene promoters). More recent studies analyzed genome-wide binding of affinity-tagged-dCas9 epigenetic editors, demonstrating high specificity both in neuroblastoma cell lines [30] and in mouse brains [54]. These findings indicate the importance of rational tool design and empirical validation to avoid potential off-target effects.

3. Special Considerations for Epigenetic Editing in Neuroscience

3.1. In Vivo Delivery Methods

Beyond the selection of the appropriate neuroepigenetic editing tool, special consideration must be given to the application of these tools in the brain, particularly with respect to delivery method. While it is possible to introduce such tools to cultured cells by biolistical particle delivery (i.e., “gene gun”) [55] and conventional transfection [33, 54, 56, 57], these methods are not suited to in vivo brain delivery. As summarized in Fig. 2, the most widely applied in vivo delivery method is stereotaxic injection of viral vectors expressing the tool of interest. Such viruses include adeno-associated virus (AAV) [58–60], characterized by long-term expression, but limited packaging capacity (~4.5 kb); Herpes Simplex Virus (HSV) [61], characterized by its short half-life (~7 days) and large packaging capacity (~14 kb); and lentivirus [59, 62–65], which allows both long-term expression and large packaging capacity but is varied in its safety in vivo due to genomic integration [59].

Fig. 2.

Brain delivery methods. (a–c) The majority of published neuroepigenetic editing studies rely on virally mediated expression of editing tool in brain. Several viral vectors are available, including HSV [31, 72, 73], AAV [29, 53, 56, 83, 100, 124], and LV [64, 75, 77, 78, 125]. HSV has emerged as the most widely applied in vivo delivery method, due to its large packaging size, neuronal specificity, and relative safety due to the lack of genomic integration. (d, e) Recent methods for nonviral delivery of purified epigenetic editing constructs. (d) Purified dCas9/sgRNA ribonucleoprotein is modified with an affinity array of nuclear localization signals to allow cell penetrance [79, 80]. In (e), a purified protein consisting of a ZFP fused to a cell-penetrating peptide gains entry to neurons [81]

Spatial specificity must be considered in the selection of delivery method, given the diversity of brain regions and neuronal and nonneuronal cell types. Furthermore, as there is meager published data on the stability of editing-induced chromatin and DNA modifications in neurons [31, 66, 67], it is crucial to consider the induction and stability of induced modification with respect to developmental time period, potential toxicity of the regulated gene, and the schedule of measured behavioral endpoints.

AAV delivery of neuroepigenetic editing tools has proved promising in preclinical models of neurodegenerative diseases. Briefly, striatal delivery of AAV-ZFP-p65 transcriptional activators targeting the endogenous glial cell line-derived neurotrophic factor (GDNF) gene rescued the deficits in a preclinical Parkinson’s disease model [56]. Studies to combat Huntington’s disease found that AAV-ZFP-KOX1 fusion was sufficient to repress expression specifically of the mutant huntingtin (HTT) gene in mice [68]. In addition to AAV-ZFPs, AAV-TALE-VP64 activators can be used to upregulate gene expression in prefrontal neurons in vivo [29]. Finally, AAV delivery of Cre recombinase and CRISPR guide RNA to a transgenic mouse expressing Cre-dependent dCas9-p300 is a new approach to epigenetic editing in vivo [60], including in brain [65]. However, the constitutively active p300 domain of dCas9 caused off-target gene activation and differential H3K27ac enrichment, even in the absence of Cre or sgRNA expression [60]. The main limitation of the AAV system in neuroepigenetic editing is its relatively small packaging limit, which is not sufficient to incorporate an expression vector containing a sgRNA and a dCas9 fused to an effector domain. The recent development of smaller Cas9 systems that are compatible with AAV [69, 70] may allow the use of this tool in neuroepigenetic editing approaches, although it still precludes the inclusion of all but the tiniest effector domains.

HSV is a delivery method used for initial studies of neuroepigenetic editing in psychiatric models, due to its neuronal specificity and large packaging size [61, 71]. HSV-ZFPs targeting the Fosb [31, 66, 72, 73] or Cdk5 [32, 74] genes have been applied to preclinical models of stress and depression. Despite the relatively short expression of 5–7 days, stereotaxic injection of HSV expressing ZFP-G9a fusions was sufficient to regulate both basal and induced gene expression, as well as regulate behavior [31]. Transient expression of Fosb-ZFP-G9a in mouse brain blocked cocaine-induced Fosb expression and reward behavior [31] and decreased aggression in mice [66]. In addition, Cre-dependent HSVs harboring a floxed stop codon upstream of the ZFP expression cassette enabled neuroepigenetic editing of the Fosb locus in a cell-type specific manner within an injected region [73], an approach critical to the elucidation of neuronal gene function (Fig. 3a).

Fig. 3.

Spatial and temporal control of epigenetic editing. (a) Cell-type specific expression of an epigenetic editing tool by injecting Cre-dependent virus into the brain of a transgenic animal expressing Cre recombinase in specific cell types [73]. (b) Light-inducible dimerization of dCas9 with a functional domain is accomplished by fusion of each to CRY2 and C1BN domains, respectively. Upon blue-light stimulation, CRY2 and C1BN heterodimerize, bringing the effector domain in proximity with the dCas9-bound locus [51]. (c) Chemically-inducible CRISPR-mediated epigenetic editing is accomplished by expression of a split-dCas9 N- and C-terminals fused to rapamycin-sensitive heterodimerization domains, FRB and FKBP, respectively. Upon rapamycin administration, dCas9 halves are brought into proximity and translocate to the nucleus [75]

Lentiviral delivery of CRISPR/Cas9 has developed substantially toward neuroepigenetic editing. Lentivirus approaches include split Cas9 [75], dCas-DNMT3a to silence APP in a model of Alzheimer’s disease [76], and lentiviral delivery of CRISPR guide RNA to a transgenic mouse expressing dCas9-p300 [60, 65]. A landmark approach applied lentiviral delivery of dCas9-Tet1 targeted to FMR1 to cultured neurons in a mouse study of Fragile X Syndrome (FXS) [67, 77]. A highly innovative lentivirus cassette co-expressed mCherry fluorescent protein, enabling fluorescence-activated cell sorting (FACS) of sgRNA-expressing cells and specific quantification of DNA methylation at the targeted genomic loci [67, 77]. Recent studies have optimized Cre-dependent lentiviral delivery of CRISPRa and CRISPRi tools in cell lines and cultured neurons [63, 64, 78].

The current neuroepigenetic editing literature primarily describes viral transduction of editing tools. However, viral vectors have limited translational application, due to the requirement of stereotaxic injection and the potential for inflammatory responses and genome perturbation. These limitations can be circumvented by the direct neuronal delivery of preassembled Cas9 ribonucleoprotein (RNP) complexes, in which Cas9 is designed with N-terminal nuclear localization signal (NLS) arrays to enable cellular uptake [79, 80]. Stereotaxic injection of 4xNLS-Cas9–2xNLS RNPs harboring a tdTomato guide RNA into a reporter mouse (Ai9) allowed direct quantification of Cas9 activity as a measure of neural tdTomato expression [79, 80]. A seminal study applied purified zinc fingers targeting the Ube3 locus in preclinical models of Angelman’s Syndrome, an imprinting neurodevelopmental disorder [81]. This study, described in further detail below, applied ZFP-VP64 fusions that contained the 10-aa transduction domain of the HIV-transactivator protein (TAT, residues 48–5719), to deliver i.p. injected, purified protein to the brain. The fusion construct specifically bound its target region as assayed by electromobility shift assay and ChIP assays in N2a cell lines, as well as activated Ube3 target gene and protein expression in mice in both hippocampus and cerebellum [81]. Recently, this approach was adapted to AAV delivery, with promising translational outcomes for Angelman’s Syndrome [82]. These encouraging results show that widespread protein activation can be accomplished in the brain in vivo using purified epigenetic editing proteins, with no apparent immunological response, garnering hopes for the eventual clinical application of this approach to neuroepigenetic editing.

3.2. Spatial and Temporal Control

Given the tremendous cellular diversity in the central nervous system, it is crucial to develop applications for cell-type-specific regulation of epigenetic editing. Brain tissue consists of thousands of intermingled cell types, each with unique gene expression profiles, connectivity, and roles in behavior. Furthermore, temporal control of epigenetic editing, ideally limited to the extent of observed endogenous effects, is necessary to elucidate the causal function of experience-dependent gene regulation. Application of inducible gene expression systems that allow spatiotemporal control of neuroepigenetic editing is emerging in neuroepigenetic editing [29, 30, 73]. Cell-type specific expression of epigenetic editing tools has been achieved through the incorporation of a loxP-stop (33polyA signal)-loxP (LSL) sequence, rendering expression inducible by Cre recombinase, which can be transgenically or virally co-expressed.

An early mouse model of Cas9 nuclease expression regulated by viral Cre delivery utilized a knock-in mouse containing Cas9 linked via a self-cleaving P2A peptide to an enhanced green fluorescent protein (EGFP) [83]. Prefrontal cortex injection of AAV-sgRNA-Cre targeting the NeuN gene led to efficient effects 3 weeks after viral transduction [83]. An additional transgenic mouse expressing dCas9-p300 suffers from nonspecific effects, as described above [60]. Recently, optimization of Cre-dependent CRISPRa with an additional intron shows results in decreased “leakiness” [78]. Recent applications of cell-type specific epigenetic editing (Fig. 3a) show success, as detailed below [72, 73].

Beyond inducible expression, inducible dimerization approaches can uncouple the expression of the DNA-binding domain (ZFPs [51], TALEs [29], and Cas9 [46, 84] from that of the transcriptional regulatory domain. This allows basal accumulation of inert components that are activated, via dimerization, with precise spatiotemporal control. Additional development of light-activated dimerization includes the combination of light-sensitive dimerizing proteins, GIGANTEA (GI) and LOV, with ZFPs for controlled dimerization in human cells [51] (Fig. 3b). Although primarily applied in nonneuronal, in vitro contexts, one study applied the CRY2 system toward light- and Cre-inducible epigenetic editing in a neuroblastoma cell line, finding robust and specific gene repression and activation using opto-dCas9-KDM1A and -p300, respectively [30]. In vitro studies found that a transient light pulse led to persistent induction of the target gene for 4 h, with expression saturating after 12 h [51], underscoring the promise of opto-epigenetic editing in neuroscience.

While light-induced dimerization is not a straightforward therapeutic approach, chemically induced dimerization addresses issues of spatiotemporal control and carries therapeutic potential. One study used a rational design strategy, based on the crystal structure of the Cas9/sgRNA/DNA complex, to split Cas9 into two fragments and added rapamycin-sensitive dimerization domains to either half [75] (Fig. 3c). Split dCas9-VP64 significantly activated expression of endogenous target genes following rapamycin treatment, although there was ~10% activation with rapamycin alone. To apply inducible dimerization to neuroepigenetic editing, additional studies must assay the temporal specificity and efficacy of these approaches in catalyzing chromatin and DNA modifications in neurons in vivo.

Consideration of the relative merits of various editing tools, delivery methods, and approaches to spatiotemporal control is critical to the application of epigenetic editing in neuroscience. Below are summarized studies that demonstrated the utility of epigenetic editing to elucidate the causal molecular function of epigenetic modifications in brain diseases.

4. Applications to Neurological Disorders

4.1. Neurodegenerative Disorders

Pharmacological modulation of DNA- or histone-modifying enzymes has shown promise in preclinical studies of neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases [85, 86]. However, small-molecule drugs broadly inhibit enzymatic activity, leading to global epigenetic and transcriptional changes. To address this limitation, several studies have applied epigenetic editing to the study of neurodegenerative disorders. An early in vivo study of Alzheimer’s disease targeted SUV39H1 to repress Psd95 expression via H3K9me enrichment [87]. Such repression was sufficient to regulate synapse and spine maturation of hippocampal neurons in vivo. One early study utilized a 6-OHDA lesion model of Parkinson’s disease, which causes the death of midbrain dopaminergic neurons and motor abnormalities [56]. The authors targeted Gdnf for epigenetic activation, since prior studies found that overexpression of GDNF protects against dopaminergic cell death in Parkinson’s disease models. Stereotaxic striatal delivery of AAV-ZFP-p65 transcriptional activators targeting Gdnf protected against loss of dopaminergic neurons as well as the behavioral sequelae [56]. More recently, dCas9-KRAB was sufficient to downregulate alpha-synuclein expression in a mouse model of Parkinson’s Disease [88]. Importantly, this study utilized a smaller molecular weight dCas9 derived from Streptococcus aureus, which allowed AAV packaging and delivery of the dCas9-KRAB fusion [88]. Finally, immune activation at the site of intracranial AAV injection was transient and resolved after six months [88]. Future epigenetic editing studies in Parkinson’s disease may build on a study of CRISPR/Cas9 deletion in Parkinson’s disease. Such gene-editing defined regulation of transcription factor binding by GWAS-identified risk variants in regulatory regions of alpha-synuclean (SCNA) [23]. Epigenetic editing can directly interrogate the sufficiency of particular transcription factors in SCNA expression and the mechanism by which enhancer hPTMs regulate such transcription factor binding.

Epigenetic editing approaches have also been applied to the study of Huntington’s disease (HD), which is characterized by mutant HTT that harbors an extended CAG leading to a polyglutamine (Q) repeat in the expressed protein. An ingenious approach to perturb mutant HTT expression examined the notion that longer CAG repeat domains contain more ZFP target sites and should thus be bound and repressed more at any given ZFP concentration [68]. This study systematically explored the binding modes of different-length ZFPs to long repetitive DNA tracts, and their relative efficacy in gene repression mediated by ZFP fused to a Kox1 domain. Kox1 recruits the co-repressor KAP1, inducing long-range repression through the spread of heterochromatin. Remarkably, in vivo, AAV-mediated delivery of the most effective HTT-ZFP-Kox1 to a HD mouse model was sufficient to reduce HTT protein aggregate, protect against a decline in motor coordination, and alleviate the clasping phenotype [68]. More recently, a study screened ZFP-KRAB to repress only the repeat-expansion HTT allele (mHTT). This approach resulted in a specific reduction in the repeat-expansion mutant HTT gene, and minimal off-target repression in human HD patient-fibroblast-derived neurons and HD model mice [89]. These key studies establish a proof-of-principle for the therapeutic potential of neuroepigenetic editing in HD, a heritable neurodegenerative disease.

Further evidence of the therapeutic potential of targeted epigenetic editing in neurodegenerative disease comes from a study of Friedreich ataxia (FRDA), a neurodegenerative and cardiac disease caused by a repressive trinucleotide (GAA) repeat expansion in the first intron of the Frataxin gene [90]. A TALE-VP64 transcriptional activator targeting the human Frataxin promoter was tested for efficacy by nucleofection into a mutant FRDA cell line. TALE-VP64 expression increased primary transcript elongation past the GAA repeat and led to higher levels of mature mRNA [90]. VP64 is a viral transcriptional activator that does not interact with the epigenome [91]. Indeed, there was no effect on transcription when TALE-VP64 was combined with inhibitors of either histone deacetylase or DNA methyltransferase. This innovative approach was then tested in fibroblasts derived from a FRDA mouse model, YG8R, containing a human mutant Frataxin knockin gene to the mouse Frataxin locus. YG8R mouse fibroblasts nucleofected with Frataxin-TALE showed increased Frataxin mRNA and protein relative to control plasmid. To advance the therapeutic potential of this approach, it will be necessary to deliver Frataxin-TALE-VP64 to all neurons and cardiomyocytes, as well as to address potential immunogenicity of the TALE fusion.

4.2. Neuropsychiatric Disorders

Chronic exposure to drugs of abuse or to stress regulates transcription factors, chromatin-modifying enzymes, and hPTMs in discrete brain regions. Furthermore, addiction and depression are highly heritable, yet it has been difficult to identify the specific genes involved, which suggests a possible role for epigenetic gene regulation in contributing to their heritability. Epigenetic regulation of Fosb, an immediate early gene, is known to underlie addiction and depression [92–94]. The first study to apply epigenetic editing in the brain in vivo targeted delivery of permissive, ZFP-p65, or repressive, ZFP-G9a, epigenetic editing to the Fosb gene [31]. Fosb-ZFP-p65 and -G9a were sufficient to modify histones H3K9/14 Ac and H3K9me2, respectively, at the targeted region of the Fosb promoter in a brain reward region, and to control drug- and stress-evoked Fosb expression in neurons and animal behavior [31]. Intriguingly, Fosb-ZFP-G9a was sufficient to block cocaine-induced Fosb activation via interference with CREB phosphorylation [31], providing direct evidence of the hierarchy between chromatin modifiers and transcription factors in gene regulation. Beyond addiction and depression, transient expression of Fosb-ZFP-G9a in the mouse brain also decreased aggression [66]. Interpretation of the persistence of these outcomes is limited because all were measured during transient expression of the epi-editor. A related study interrogated epi-editing of the immediate early gene, Fos. Targeted Fos enhancer acetylation with dCas9-p300 increased endogenous neuronal physiology and signaling [60]. However, interpretation of these results is limited regarding (1) the persistence of the effect, due to AAV delivery of sgRNA and (2) specificity, due to the off-target activity of constitutively expressed dCas9-p300 (as described above).

Additional studies of Fosb applied cell-type specific epigenetic editing in brain. In one study, stress susceptibility was oppositely regulated by distinct specific cell types and histone modifications [73], as predicted from the cell-type specific expression Fosb following stress [95]. Importantly, neuronal-subtype specific, transient expression of Fosb-ZFP-G9a was sufficient to promote depressive-like behavior for at least four days beyond Fosb-ZFP-G9a epi-editor expression [73]. Finally, activation of Fosb with dCas9-CREB defined cell-type specific, Fosb transcriptional regulation in the absence of drug or stress exposure [72]. These studies underscore the utility of ZFP-based epigenetic editing to model naturally occurring transcriptional phenomena that control behavior.

Fosb is just one of thousands of genes under epigenetic control in the context of stress and drug exposure. One target of Fosb is Cdk5, a neuron-specific kinase implicated in reward processing, stress, and memory. Epigenetic editing using Cdk5-ZFP-p65 and Cdk5-ZFP-G9a has elucidated the epigenetic regulation of Cdk5 in mouse models of post-traumatic stress disorder (PTSD) [74], cocaine reward, and stress [32]. Specifically, Cdk5-targeted H3K9/14ac increased cocaine-induced locomotor behavior, as well as resilience to social stress. Conversely, Cdk5-targeted H3K9me2 attenuated cocaine reward but had no effect on stress-induced behavior. These data are especially compelling given that conventional Cdk5 overexpression or knockdown caused opposite behavioral phenotypes [96–98]. A more recent study of epigenetic regulation of Cdk5 in the context of diabetes-associated development of AD supports the translational relevance of epigenetic editing of Cdk5 [99]. Finally, a study of alcohol use disorder targeted dCas9-p300 to an enhancer of key immediate early gene, Arc [65]. dCas9-p300 increased Arc mRNA, Arc promoter H3K27Ac, Arc eRNA, and the interaction strength between Arc enhancer and promoter, rescuing the behavioral effects of alcohol exposure [65].

4.3. Neurodevelopmental Disorders

Models of neurodevelopmental diseases have been developed by epigenetic editing in early development. Epigenetic editing in cultured cerebellar granule cells with dCas9-VP64 and sgRNAs targeting either of two developmentally regulated Grin2c putative enhancers sites was sufficient to specifically activate Grin2c expression [26]. Neurodevelopmental disorders were also studied using CRISPR/Cas9 gene editing, in studies of autism spectrum disorder (ASD), for example [53, 100, 101]. Targeted de/methylation using CRISPR tools have been applied to imprinting disease [102], autism [103], neurodevelopmental disorders [104], and Silver Russel syndrome [105], as reviewed elsewhere [106].

Two studies of epigenetic editing of DNA methylation applied ex vivo transplantation to rescue neurodevelopmental diseases, Rett’s Syndrome and Fragile X Syndrome (FXS) [67, 107]. These diseases are marked by epigenetic dysregulation of key developmental genes, namely, MECP2 and FMR1, respectively, and preclinical studies show promise of epigenetic editing therein. Lentiviral delivery of dCas9-Tet1 or dCas-Dnmt3 induced demethylation or de novo methylation, respectively, at target genes in cultured mouse cortical neurons. Brain-derived neurotrophic factor (BDNF) expression levels were induced, and cytosine hydroxymethylation was detected in cultured neurons following dCas9-Tet1 + BDNF-sgRNA expression, as quantified in cells obtained by FACS of sgRNA-infected cells co-expressing mCherry fluorescent protein [77]. The utility of this approach was validated with lentiviral dCas9-Tet1 and Bdnf-sgRNA delivery to a transgenic mouse containing a methylation-sensitive GFP reporter, suggesting that the viral delivery of dCas9-Tet1 and sgRNA may be viable for CRISPR/dCas9 targeted DNA methylation in rodent brain [77]. In a follow-up study, an FXS mouse model phenotype, in which FMR1 is repressed, was rescued by transplantation of ex vivo iPSCs epi-edited with dCas9-Tet1 targeted to FMR1 [67]. Importantly, epi-editing was sufficient to activate FMR1 expression in postmitotic neurons cultured from the FXS mouse model, despite the fact that FMR1 with repeat expansion is heterochromatic and transcriptionally silenced. Furthermore, persistent lentiviral expression of dCas9-Tet1 and sgRNAs in embryonic cells was sufficient for sustained FMR1 activation in neurons even up to three months posttransplantation. Such persistent unsilencing demonstrates that endogenous mechanisms of FMR1 repression did not override the epi-editing by dCas9-Tet1 [67]. This promising approach suggests future research to establish epigenetic editing of FMR1 in vivo.

A related study applied epigenetic editing to human ESCs derived from Rett Syndrome patients, in which one allele of X-linked MECP2 is mutated [107]. dCas9-Tet1 targeted to wild-type MECP2 on the inactivated X chromosome is sufficient to demethylate and activate MECP2 expression and rescue deficits in hESC-derived neurons [107]. Although the latter study did not transplant the epi-edited neurons to a mouse model of Rett Syndrome, these studies underscore the enormous therapeutic potential of epi-editing in neurodevelopmental disease. Finally, studies of Angelman’s Syndrome mark a major breakthrough in epigenetic editing toward neurodevelopmental disease, as described above [81, 82].

5. Applications to Basic Neuroscience

Locus-specific epigenetic editing allows the experimental discrimination between the mere presence and functional relevance of chromatin and DNA modifications. Several studies have utilized neuroepigenetic editing to decipher the causal role of DNA methylation in neuronal gene regulation. Interestingly, an early study utilizing ZFPs in cell lines found improved target specificity when a prokaryotic DNA methyltransferase was mutated to a less active form, reducing spurious genome-wide methylation [108]. Similarly to the endogenous mechanisms of methylated DNA-mediated gene repression, ZFP-initiated DNA methylation led to enrichment of H3K9me2 and depletion of H3K4me3, which may explain the reported inheritance of the induced methylation in this case. Furthermore, the methylation status persisted beyond expression of the ZFP, indicating that targeted DNA methylation can be exploited for sustained gene repression [108]. However, one limitation of this approach was the use of an integrated, rather than endogenous, target site. In fact, in another investigation, which targeted a DNA methyltransferase and the histone methyltransferase GLP to the VEGF gene in a human cell line found that, upon loss of the targeted methyltransferases, the induced epigenetic marks (in this case, DNA methylation and H3K9me2) returned to baseline, indicating that the methylation was not stable during cell division [109]. Differing results from additional studies [110, 111] suggest that cell line, gene target and delivery method contribute to the stability, and inheritance of induced epigenetic modifications. While one study suggests that the behavioral effects of histone methylation in vivo can persist beyond expression of the transgene [31], a systematic investigation is necessary to determine the persistence of epigenetic editing in neurons in vivo, especially considering that these cells are generally nondividing.

CRISPR/Cas9 gene editing (e.g., gene knockout via indel formation) has been applied to neurons in vivo, demonstrating the potential of CRISPR/dCas9 in neuroepigenetic editing. An innovative dual AAV system was used to target methyl CpG binding protein (Mecp2) mutations that underlie Rett syndrome, a neurodevelopmental disease, with Cas9 and sgRNA under the control of the neuronal Mecp2 and Synapsin 1 promoters, respectively [53]. As referenced above, co-expression of sgRNA with KASH-GFP allows FACS of infected neurons by driving GFP to the outer nuclear membrane; in this case, over 70% of primary cortical neurons showed loss of Mecp2 expression when Cas9, but not dCas9, was co-injected [53]. To validate the potential utility of CRISPR/Cas9 gene editing in specific brain regions, AAV-sgRNA and AAV-Cas9 were injected into mouse hippocampus. Mecp2 indels (induced mutations) were found in 70% of FACS-sorted nuclei, while Mecp2 protein levels were reduced by 60%.

Of particular interest in basic neurobiology is epigenetic regulation of neuronal enhancers. The immediate early gene, c-Fos, is regulated by several enhancers scattered around the gene, yet little is known about its coordinated regulation in response to stimulation. To address this, one group defined activity-dependent c-Fos enhancers based on activity-induced enrichment of specific hPTMs and transcription factors as well as induction of enhancer RNAs [112]. Activity-dependent c-Fos enhancers were then individually targeted with dCas9-KRAB, and the activity-specific induction of c-Fos from each enhancer was quantified by enhancer RNA and mRNA expression [112]. This study, which highlights the promising utility of neuroepigenetic editing to define molecular mechanisms of stimulus-induced gene expression, uncovered that BDNF-induced c-Fos expression is controlled by a subset of coordinated, activity-dependent c-Fos enhancers [112]. This and other studies underscore the utility of epigenetic editing to functionally validate enhancer elements, a critically important application of epigenetic editing both in the brain [65] and other tissues [20, 44]. Finally, as mentioned above, DNAse hypersensitive site (DHS) mapping and RNA-sequencing were combined to identify developmentally regulated promoter and enhancer elements, whose function was predicted based on the presence of specific hPTMs (e.g., H3K27me2) [26]. To directly test the causal relevance of DHS opening in enhancer function, dCas9-VP64 was targeted to enhancers of Grin2c, which encodes a developmentally regulated NMDA-type glutamate receptor subunit that mediates mature synaptic function.

Finally, while chromatin regulation of gene expression is well established, recent studies find that certain epigenetic modifications are strongly predictive of alternative splicing. A key study in mouse brain applied exon-specific epigenetic editing of H3K36me3 using dCas9-SET2 to drive alternative splicing of Srsf11, which regulated mouse cocaine reward behavior [54]. A related study found that exon-targeted demethylation with dCas9-TET1 in a neuroblastoma cell line increased alternative exon inclusion of the calcium channel gene, Cacna1b [113]. Importantly, these studies demonstrated that epigenetic regulation of alternative splicing can be independent of gene expression.

6. Future Applications: Combinatorial Approaches

Gene expression profiling studies in the brain have revealed that complex regulatory networks of mRNA expression underlie normal brain function, development, and neuropsychiatric disease [6, 14, 114]. Thus, attempts to control neuronal function and behavior by epigenetic editing of a single locus may be limited in their application. Recent advances in combinatorial approaches to epigenetic editing have been applied to numerous types of epigenetic modifications. For example, Cas9 can be combined with multiple sgRNAs [42] to achieve efficient multiplexed genome editing in mammalian cells [115, 116]. In general, two main approaches to multiplexing are apparent: those involving multiple effector domains and those involving multiple gene targets.

Comprehensive analyses of both approaches in yeast and mammalian cell lines reveal potential applications in neuroscience. For example, an exhaustive quantitative study of 223 yeast chromatin regulators fused to ZFPs and targeted to a synthetic transcriptional reporter found that the precise location of ZFP binding respective to the TSS was a determining factor in its efficacy, as measured by gene expression and histone modification [117]. Reporter gene activity revealed that activation domains generally function when recruited to specific locations (e.g., promoters and enhancers), while repression is controlled throughout a gene, perhaps through heterochromatin spreading mechanisms or disruption of transcription elongation. The observed regulatory hierarchy informs the future selection of loci targeted for combinatorial regulation, given that a single site may have differential effects on repression or activation based on its location relative to specific gene elements.

The combination of multiple effectors has also been applied to mimic the natural cooperative recruitment process inherent to gene activation. To test whether functional domains fused in tandem would increase transcriptional activation, one study fused dCas9 to a series of more than 20 functional domains, and assessed their potency in activating a fluorescent reporter in HEK293 cells [118]. Additionally, a successful and versatile design for the co-recruitment of functional domains is that of dCas9 fused to a repeating peptide array termed SunTag, a protein scaffold that recruits multiple copies of an antibody-functional domain fusion [119]. Comparison of the efficacy of lentivirus expression of dCas9-VP64 and dCas9-SunTag-VP64 in cell culture found that recruitment of multiple VP64 proteins via the SunTag potentiated VP64 mediated activation [119]. The result is extremely promising given the versatility of the antibody-based SunTag design. Such multiplexing approaches identified tandem recruitment arrangements that could activate genes between 280 and 20,000 times relative to control [118, 119], which may be useful in certain applications (e.g., iPSC differentiation) but undermines the goal of epigenetic editing, stated earlier, to recapitulate physiologically relevant levels and modes of gene regulation. Targeting of multiple effector domains is well established in non-neuronal systems [120–122]. Recent studies have found that combinatorial approaches to targeted repression are more robust and sustained, compared to targeting of individual moieties. A recent system, CRISPRoff induced sustained repression with dCas9, fused to both KRAB and DNMT3A3L [123].

Beyond combinatorial effector domains, simultaneous epigenetic editing of multiple target loci has great potential utility for gene network regulation in the brain [8, 14]. Although multiplexed epigenetic editing has not yet been accomplished in neurons, multiplexed CRISPR/Cas9 gene knockout was used to investigate the functional redundancy of DNA methyltransferases. In this study, a cocktail of AAV-Cas9 and AAV-sgRNAs targeting the Dnmt3a, Dnmt1, and Dnmt3b gene loci was stereotaxically injected into the hippocampus of adult mice [53]. GFP-KASH FACS and next-generation DNA sequencing found that approximately 62% of all transduced neurons contained indels in both Dnmt1 and Dnmt3a, compared to ~35% which contained simultaneous modification of Dnmt1, Dnmt3a, and Dnmt3b [53]. Additional in vivo multiplexing studies have targeted lung epithelial tissue with a single AAV vector containing sgRNAs targeting multiple tumor suppressor genes [83], as well as combinatorial methods to drive cell differentiation [46, 84]. The results of these studies underscore the challenge of multiplexed gene targeting in vivo and remain a potential source of inspiration for approaches to combinatorial neuroepigenetic editing.

7. Conclusion

Experience and learning regulate genome-wide deposition of hPTMs and expression of enzymes that catalyze and metabolize them. Herculean efforts in next-generation ChIP- and RNA-sequencing of brain regions have convinced the field that, indeed, regulation of the epigenetic landscape contributes to neuronal development and function. Furthermore, precise spatiotemporal transcriptional control is crucial to the highly specialized role of neurons in integrating external stimuli. However, major questions remain about the functional role played by many of the epigenetic modifications observed to date in brain. That is, given the promiscuity of the enzymes involved, it is challenging to obtain direct causal evidence of the function of epigenetic remodeling that occurs at a single gene in a single cell type within a given brain region of interest in awake, behaving animals. Neuroepigenetic editing methods are poised to address this limitation and, while this field is in its relative infancy, great strides have been made to directly test the functional role of specific histone and DNA modifications in the mammalian brain in vivo. This work is central to delineate a precise understanding of epigenetic regulation in the brain as well as perhaps to one day devise targeted epigenetic therapies for neuropsychiatric illness.