Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

In vivo locus-specific editing of the neuroepigenome


Studies over the past several decades have identified numerous epigenetic mechanisms associated with pathological states in psychiatric and neurological disease. Until recently, studies investigating chromatin-regulatory proteins, using overexpression or knockdown approaches, did not establish causal roles for epigenetic modifications at specific genes because these techniques typically affect hundreds or thousands of genomic loci. In this Review, we describe recent efforts in using locus-specific neuroepigenome editing in vivo to, for the first time, define causal relationships between a single chromatin modification at a specific gene in a defined cell population and downstream measures at the molecular, cellular, circuit and behavioural levels. We briefly introduce three epigenome-editing platforms: zinc-finger proteins, transcriptional activator-like effectors and clustered regularly interspaced short palindromic repeats (CRISPR). We then explore the development of in vivo neuroepigenome-editing tools and their applications to resolve epigenetic contributions to the pathophysiology of brain diseases. We also discuss technical considerations for in vivo neuroepigenome-editing experiments and ongoing innovations in the field, including new tools to investigate chromatin marks, manipulate chromatin topology and induce epigenetic modifications at multiple genes in the same cell. Lastly, we explore the potential clinical applications of in vivo neuroepigenome editing for treating brain pathology.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: In vivo neuroepigenome-editing tools.
Fig. 2: Overview of CRISPRa/CRISPRi techniques.
Fig. 3: CRISPR-based neuroepigenome-editing tools.
Fig. 4: Innovative neuroepigenome-editing tools.


  1. 1.

    Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001).

    CAS  PubMed  Google Scholar 

  2. 2.

    Suzuki, M. M. & Bird, A. DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9, 465–476 (2008).

    CAS  PubMed  Google Scholar 

  3. 3.

    Mercer, T. R. & Mattick, J. S. Structure and function of long noncoding RNAs in epigenetic regulation. Nat. Struct. Mol. Biol. 20, 300–307 (2013).

    CAS  PubMed  Google Scholar 

  4. 4.

    Liu, X. S. & Jaenisch, R. Editing the epigenome to tackle brain disorders. Trends Neurosci. 42, 861–870 (2019).

    CAS  PubMed  Google Scholar 

  5. 5.

    Fellmann, C., Gowen, B. G., Lin, P. C., Doudna, J. A. & Corn, J. E. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nat. Rev. Drug. Discov. 16, 89–100 (2017).

    CAS  PubMed  Google Scholar 

  6. 6.

    Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Dreier, B., Beerli, R. R., Segal, D. J., Flippin, J. D. & Barbas, C. F. 3rd Development of zinc finger domains for recognition of the 5’-ANN-3’ family of DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 276, 29466–29478 (2001).

    CAS  PubMed  Google Scholar 

  9. 9.

    Gilbert, L. A. et al. CRISPR-Mediated Modular RNA-guided regulation of transcription in Eukaryotes. Cell 154, 442–451 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Lorsch, Z. S. et al. Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex. Nat. Neurosci. 22, 1413–1423 (2019). Neuroepigenome editing with CRISPR is used to direct CREB to the Zfp189 promoter (a CREB target) in PFC neurons. The resulting induction of endogenous Zfp189 promotes resilience to behavioural stress.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Cates, H. M. et al. Transcription factor E2F3a in nucleus accumbens affects cocaine action via transcription and alternative splicing. Biol. Psychiatry 84, 167–179 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Zhou, H. B. et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice. Nat. Neurosci. 21, 440–446 (2018).

    CAS  PubMed  Google Scholar 

  13. 13.

    Savell, K. E. et al. A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation. eNeuro (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Peter, C. J. et al. In vivo epigenetic editing of Sema6a promoter reverses transcallosal dysconnectivity caused by C11orf46/Arl14ep risk gene. Nat. Commun. 10, 4112 (2019).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Lu, Z. et al. Locus-specific DNA methylation of Mecp2 promoter leads to autism-like phenotypes in mice. Cell Death Dis. 11, 85 (2020). DNA methylation of the Mecp2 promoter in the hippocampus is achieved by targeting dCas9 fused to a DNMT catalytic domain to the locus. This action suppresses Mecp2 expression and is sufficient to induce autism-like behavioural phenotypes.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Klug, A. & Rhodes, D. Zinc fingers: a novel protein fold for nucleic-acid recognition. Cold Spring Harb Symp. Quant. Biol. 52, 473–482 (1987).

    CAS  PubMed  Google Scholar 

  17. 17.

    Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).

    CAS  PubMed  Google Scholar 

  18. 18.

    Camenisch, T. D., Brilliant, M. H. & Segal, D. J. Critical parameters for genome editing using zinc finger nucleases. Mini Rev. Med. Chem. 8, 669–676 (2008).

    CAS  PubMed  Google Scholar 

  19. 19.

    Gordley, R. M., Smith, J. D., Graslund, T. & Barbas, C. F. III. Evolution of programmable zinc finger-recombinases with activity in human cells. J. Mol. Biol. 367, 802–813 (2007).

    CAS  PubMed  Google Scholar 

  20. 20.

    Kolb, A. F. et al. Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction. Trends Biotechnol. 23, 399–406 (2005).

    CAS  PubMed  Google Scholar 

  21. 21.

    Laganiere, J. et al. An engineered zinc finger protein activator of the endogenous glial cell line-derived neurotrophic factor gene provides functional neuroprotection in a rat model of Parkinson’s disease. J. Neurosci. 30, 16469–16474 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Bustos, F. J. et al. Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer’s disease mice. Brain 140, 3252–3268 (2017).

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Heller, E. A. et al. Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors. Nat. Neurosci. 17, 1720–1727 (2014). ZFPs fused to p65 or to G9a are targeted to the Fosb locus in NAc neurons; they bidirectionally control Fosb expression as well as downstream behavioural responses to social stress or cocaine.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Lobo, M. K. et al. ΔFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli. J. Neurosci. 33, 18381–18395 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Robison, A. J. & Nestler, E. J. Transcriptional and epigenetic mechanisms of addiction. Nat. Rev. Neurosci. 12, 623–637 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Maze, I. et al. Essential role of the histone methyltransferase G9a in cocaine-induced plasticity. Science 327, 213–216 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Renthal, W. et al. Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins. Neuron 62, 335–348 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Kumar, A. et al. Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron 48, 303–314 (2005).

    CAS  PubMed  Google Scholar 

  29. 29.

    Covington, H. E. III et al. A role for repressive histone methylation in cocaine-induced vulnerability to stress. Neuron 71, 656–670 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Aleyasin, H. et al. Cell-type-specific role of ΔFosB in nucleus accumbens in modulating intermale aggression. J. Neurosci. 38, 5913–5924 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Hamilton, P. J. et al. Cell-type-specific epigenetic editing at the Fosb gene controls susceptibility to social defeat stress. Neuropsychopharmacology 43, 272–284 (2018). ZFPs developed by Heller et al. (2014) are expressed in the D1R-expressing or D2R-expressing MSNs in the NAc, where they are shown to produce opposite effects on depression-like behaviours.

    CAS  PubMed  Google Scholar 

  32. 32.

    Heller, E. A. et al. Targeted epigenetic remodeling of the Cdk5 gene in nucleus accumbens regulates cocaine- and stress-evoked behavior. J. Neurosci. 36, 4690–4697 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Su, S. C., Rudenko, A., Cho, S. & Tsai, L. H. Forebrain-specific deletion of Cdk5 in pyramidal neurons results in mania-like behavior and cognitive impairment. Neurobiol. Learn. Mem. 105, 54–62 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Hawasli, A. H. et al. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nat. Neurosci. 10, 880–886 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Zhong, P. et al. Cyclin-dependent kinase 5 in the ventral tegmental area regulates depression-related behaviors. J. Neurosci. 34, 6352–6366 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Sase, A. S. et al. Sex-specific regulation of fear memory by targeted epigenetic editing of Cdk5. Biol. Psychiatry 85, 623–634 (2019).

    CAS  PubMed  Google Scholar 

  37. 37.

    Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501–1501 (2009).

    CAS  PubMed  Google Scholar 

  38. 38.

    Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Toegel, M. et al. A multiplexable TALE-based binary expression system for in vivo cellular interaction studies. Nat. Commun. 8, 1663 (2017).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011).

    CAS  PubMed  Google Scholar 

  42. 42.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Heidenreich, M. & Zhang, F. Applications of CRISPR-Cas systems in neuroscience. Nat. Rev. Neurosci. 17, 36–44 (2016).

    CAS  PubMed  Google Scholar 

  45. 45.

    Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

    CAS  PubMed  Google Scholar 

  47. 47.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Carpenter, M. D. et al. Nr4a1 suppresses cocaine-induced behavior via epigenetic regulation of homeostatic target genes. Nat. Commun. 11, 504 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Zheng, Y. et al. CRISPR interference-based specific and efficient gene inactivation in the brain. Nat. Neurosci. 21, 447–454 (2018).

    CAS  PubMed  Google Scholar 

  50. 50.

    Devesa-Guerra, I. et al. DNA methylation editing by CRISPR-guided excision of 5-methylcytosine. J. Mol. Biol. 432, 2204–2216 (2020).

    CAS  PubMed  Google Scholar 

  51. 51.

    Engmann, O. et al. Cocaine-induced chromatin modifications associate with increased expression and three-dimensional looping of Auts2. Biol. Psychiatry 82, 794–805 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247 e217 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017).

    CAS  PubMed  Google Scholar 

  55. 55.

    Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992 e976 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Kwon, D. Y., Zhao, Y. T., Lamonica, J. M. & Zhou, Z. Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC. Nat. Commun. 8, 15315 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat. Methods 12, 401–403 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Vojta, A. et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Chen, L. F. et al. Enhancer histone acetylation modulates transcriptional bursting dynamics of neuronal activity-inducible genes. Cell Rep. 26, 1174–1188 e1175 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Benavides, D. R. et al. Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability. J. Neurosci. 27, 12967–12976 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Massart, R. et al. Role of DNA methylation in the nucleus accumbens in incubation of cocaine craving. J. Neurosci. 35, 8042–8058 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Klengel, T., Pape, J., Binder, E. B. & Mehta, D. The role of DNA methylation in stress-related psychiatric disorders. Neuropharmacology 80, 115–132 (2014).

    CAS  PubMed  Google Scholar 

  65. 65.

    Al-Mahdawi, S., Virmouni, S. A. & Pook, M. A. The emerging role of 5-hydroxymethylcytosine in neurodegenerative diseases. Front. Neurosci. 8, 397 (2014).

    PubMed  PubMed Central  Google Scholar 

  66. 66.

    McClung, C. A. & Nestler, E. J. Regulation of gene expression and cocaine reward by CREB and ΔFosB. Nat. Neurosci. 6, 1208–1215 (2003).

    CAS  PubMed  Google Scholar 

  67. 67.

    Lardner, C. K. et al. in Neuroscience 2019 Vol. Program Number 415.17 (Society for Neuroscience, 2019).

  68. 68.

    Najmabadi, H. et al. Deep sequencing reveals 50 novel genes for recessive cognitive disorders. Nature 478, 57–63 (2011).

    CAS  PubMed  Google Scholar 

  69. 69.

    Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015). A novel CRISPRa/CRISPRi approach is used to recruit distinct transcriptional effector domains to separate genes within the same cell.

    CAS  PubMed  Google Scholar 

  70. 70.

    Ofengeim, D., Giagtzoglou, N., Huh, D., Zou, C. & Yuan, J. Single-cell RNA sequencing: unraveling the brain one cell at a time. Trends Mol. Med. 23, 563–576 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Back, S. et al. Neuron-specific genome modification in the adult rat brain using CRISPR-Cas9 transgenic rats. Neuron 102, 105–119 (2019).

    CAS  PubMed  Google Scholar 

  72. 72.

    Daigle, T. L. et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465–480 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Kugler, S., Kilic, E. & Bahr, M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347 (2003).

    CAS  PubMed  Google Scholar 

  74. 74.

    Shevtsova, Z., Malik, J. M., Michel, U., Bahr, M. & Kugler, S. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp. Physiol. 90, 53–59 (2005).

    CAS  PubMed  Google Scholar 

  75. 75.

    Merienne, N., Le Douce, J., Faivre, E., Deglon, N. & Bonvento, G. Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors. Front. Cell Neurosci. 7, 106 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Blankvoort, S., Descamps, L. A. L. & Kentros, C. Enhancer-driven gene expression (EDGE) enables the generation of cell type specific tools for the analysis of neural circuits. Neurosci. Res. 152, 78–86 (2020).

    CAS  PubMed  Google Scholar 

  77. 77.

    Morgan, S. L. et al. Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping. Nat. Commun. 8, 15993 (2017). CRISPR tools are used to chemically induce, in a reversible manner, the chromatin looping between two genes.

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Wang, H. et al. CRISPR-mediated programmable 3D genome positioning and nuclear organization. Cell 175, 1405–1417 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Day, J. J. Genetic and epigenetic editing in nervous system. Dialogues Clin. Neurosci. 21, 359–368 (2019).

    PubMed  PubMed Central  Google Scholar 

  80. 80.

    Hamilton, P. J., Lim, C. J., Nestler, E. J. & Heller, E. A. Neuroepigenetic editing. Methods Mol. Biol. 1767, 113–136 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Carlezon, W. A. Jr., Nestler, E. J. & Neve, R. L. Herpes simplex virus-mediated gene transfer as a tool for neuropsychiatric research. Crit. Rev. Neurobiol. 14, 47–67 (2000).

    CAS  PubMed  Google Scholar 

  82. 82.

    Nelson, C. E. & Gersbach, C. A. Engineering delivery vehicles for genome editing. Annu. Rev. Chem. Biomol. Eng. 7, 637–662 (2016).

    CAS  PubMed  Google Scholar 

  83. 83.

    Thakore, P. I. et al. RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors. Nat. Commun. 9, 1674 (2018).

    PubMed  PubMed Central  Google Scholar 

  84. 84.

    Vora, S. et al. Rational design of a compact CRISPR-Cas9 activator for AAV-mediated delivery. bioRxiv (2018).

    Article  Google Scholar 

  85. 85.

    Maeder, M. L. & Gersbach, C. A. Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430–446 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Ren, J. & Zhao, Y. Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9. Protein Cell 8, 634–643 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 88.

    Wood, E. H. et al. Stem cell therapies, gene-based therapies, optogenetics, and retinal prosthetics: current state and implications for the future. Retina 39, 820–835 (2019).

    PubMed  PubMed Central  Google Scholar 

  89. 89.

    Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu. Rev. Virol. 4, 37–59 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Marino, N. D., Pinilla-Redondo, R., Csorgo, B. & Bondy-Denomy, J. Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Nat. Methods 17, 471–479 (2020).

    CAS  PubMed  Google Scholar 

  91. 91.

    Pawluk, A., Davidson, A. R. & Maxwell, K. L. Anti-CRISPR: discovery, mechanism and function. Nat. Rev. Microbiol. 16, 12–17 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. 93.

    Stavarache, M. A. et al. Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound. J. Neurosurg. 130, 989–998 (2018).

    PubMed  Google Scholar 

  94. 94.

    Lee, B. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat. Biomed. Eng. 2, 497–507 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. 95.

    Moreno, A. M. et al. Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy. Nat. Biomed. Eng. 3, 806–816 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 96.

    Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. 97.

    Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    CAS  PubMed  Google Scholar 

  98. 98.

    Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. 99.

    Liu, Y. et al. CRISPR activation screens systematically identify factors that drive neuronal fate and reprogramming. Cell Stem Cell 23, 758–771 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. 100.

    Jin, X. et al. In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes. bioRxiv (2019).

    Article  Google Scholar 

  101. 101.

    Thyme, S. B. et al. Phenotypic landscape of schizophrenia-associated genes defines candidates and their shared functions. Cell 177, 478–491 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 102.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. 103.

    Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015). A light-inducible CRISPRa/CRISPRi approach using heterodimerization domains tethered to dCas9 and the transcriptional effector domain is developed.

    CAS  PubMed  PubMed Central  Google Scholar 

  104. 104.

    Farrelly, L. A. et al. Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature 567, 535–539 (2019). Serotonylation at histone H3 glutamine 5, in close proximity to the well-characterized histone H3 lysine 4 trimethylation mark, was shown to regulate gene expression in the brain.

    CAS  PubMed  PubMed Central  Google Scholar 

  105. 105.

    Meers, M. P., Bryson, T. D., Henikoff, J. G. & Henikoff, S. Improved CUT&RUN chromatin profiling tools. eLife (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107.

    Kocak, D. D. et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat. Biotechnol. 37, 657–666 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


The authors are very grateful to J. K. Gregory for her help with the figures in this Review.

Author information




E.J.N. made substantial contributions to the discussion and editing of the content. Y.YY. and C.D.T. wrote the article. All authors reviewed and edited the article before submission.

Corresponding author

Correspondence to Eric J. Nestler.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information

Nature Reviews Neuroscience thanks J. Day, H. Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Transcription activator-like effector

(TALE). DNA-binding protein derived from bacteria (Xanthomonas) that regulates gene expression.

Clustered regularly interspaced short palindromic repeats

(CRISPR). A component of the adaptive immune system in bacteria and archaea that cleaves foreign nucleic acid sequences. It is used routinely in the laboratory to enable targeted genetic and epigenetic manipulations.

Guide RNA

(gRNA). A synthetic RNA that guides clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) to a specific DNA sequence in the genome.


A complex of four copies of VP16 (a viral protein sequence of 16 amino acids) that activates gene transcription.

Zinc-finger proteins

(ZFPs). Proteins consisting of zinc ion-regulated Cys2-His2 domains that recognize specific 18-bp sequences of DNA. These proteins can be fused to various effector proteins, including nucleases and chromatin-modifying proteins.

Transcription factor

(TF). Protein that binds to specific sequences of DNA and regulates gene expression through the recruitment of chromatin-modifying enzymes and other proteins.


An immediate early gene that encodes full-length FOSB and a truncated splice variant ∆FOSB, and that has served as a useful target for the development of novel neuroepigenome-editing tools.

cAMP response element-binding protein

(CREB). A ubiquitously expressed transcription factor implicated in diverse functions in the central nervous system and periphery.

Protospacer adjacent motif

(PAM). A short DNA sequence upstream of the target gene that is recognized by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9).

CRISPR activation

(CRISPRa). A clustered regularly interspaced short palindromic repeats (CRISPR) system that uses potent activation domains, such as the viral transcription factor VP64, to increase gene expression.

CRISPR interference

(CRISPRi). A clustered regularly interspaced short palindromic repeats (CRISPR) system that uses repressive domains, such as the Krüppel-associated box (KRAB) domain, to suppress gene expression.


A clustered regularly interspaced short palindromic repeats (CRISPR)-based method that uses a repeating peptide array to recruit multiple copies of single-chain variable fragment (scFv)-fused effector proteins to a target gene.


A putative transcription factor whose gene is a target of cAMP response element-binding protein (CREB). Recent studies suggest that this protein is involved in regulating synaptic plasticity and behavioural responses to stress.


Enzyme that catalyses the addition of methyl groups to DNA.

Chromatin loop reorganization using CRISPR–dCas9

(CLOuD9). A clustered regularly interspaced short palindromic repeats (CRISPR) system that uses chemically induced ligation to selectively and reversibly establish chromatin loops.

CRISPR genome organization

(CRISPR-GO). A clustered regularly interspaced short palindromic repeats (CRISPR) system that uses chemically induced ligation to bring loci in close proximity to nuclear subcompartments.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yim, Y.Y., Teague, C.D. & Nestler, E.J. In vivo locus-specific editing of the neuroepigenome. Nat Rev Neurosci 21, 471–484 (2020).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing