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CRISPR activation for SCN2A-related neurodevelopmental disorders

Nature volume 646, pages 983–991 (2025)Cite this article

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A Publisher Correction to this article was published on 08 December 2025

This article has been updated

Abstract

Most neurodevelopmental disorders with single gene diagnoses act via haploinsufficiency, in which only one of the two gene copies is functional1. SCN2A haploinsufficiency is one of the most frequent causes of neurodevelopmental disorder, often presenting with autism spectrum disorder, intellectual disability and, in a subset of children, refractory epilepsy2. Here, using SCN2A haploinsufficiency as a proof-of-concept, we show that upregulation of the existing functional gene copy through CRISPR activation (CRISPRa) can rescue neurological-associated phenotypes in Scn2a haploinsufficient mice. We first show that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency (Scn2a+/−). Next, using an adeno-associated virus CRISPRa-based treatment in adolescent mice, we show that we can correct intrinsic and synaptic deficits in neocortical pyramidal cells, a major cell type that contributes to neurodevelopmental disorders and seizure aetiology in SCN2A haploinsufficiency. Furthermore, we find that systemic delivery of CRISPRa protects Scn2a+/− mice against chemoconvulsant-induced seizures. Finally, we also show that adeno-associated virus CRISPRa treatment rescues excitability in SCN2A haploinsufficient human stem-cell-derived neurons. Our results showcase the potential of this therapeutic approach to rescue SCN2A haploinsufficiency and demonstrates that rescue even at adolescent stages can ameliorate neurodevelopmental phenotypes.

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Fig. 1: Genetic rescue of Scn2a haploinsufficiency in adolescent mice rescues electrophysiological deficits.
Fig. 2: Intracranial Scn2a-rAAV-CRISPRa in mPFC rescues excitability deficits in Scn2a+/− neurons.
Fig. 3: Systemic CRT rescues electrophysiological deficits and is protective against chemoconvulsant-induced seizure.
Fig. 4: SCN2A-rAAV-CRISPRa rescues spiking properties of SCN2A+/− hESC-derived neurons.

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Data availability

All RNA-seq data are available at the NCBI Gene Expression Omnibus as Bioproject GSE302897. Requests for materials can be directed to N.A. (CRT-based approaches) or K.J.B. (mouse models). Source data are provided with this paper.

Code availability

No custom code was generated for this work.

Change history

  • 23 February 2026

    In the version of the article initially published, in the “CRISPRa in vitro optimization” section of the Methods, the text “rAAV vectors were generated using similar plasmids and cloning methods as described previously” originally cited ref. 1 but has now been corrected to ref. 17 in the HTML and PDF versions of the article.

  • 08 December 2025

    A Correction to this paper has been published: https://doi.org/10.1038/s41586-025-09995-9

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Acknowledgements

We thank the Bender and Ahituv laboratory members for comments and discussions on this manuscript, and members of the FamilieSCN2A Foundation who provided the core motivation and inspiration for this work. We acknowledge the UCSF Parnassus Flow CoLab (RRID:SCR_018206) supported in part by NIH grant no. P30 DK063720 and by the NIH S10 Instrumentation Grant no. S10 1S10OD021822-01. This work was supported by grants from SFARI (grant no. 629287: K.J.B., N.A.; grant no. 513133: K.J.B.), the Broad Institute Target Practice Initiative (K.J.B.), the Autism Science Foundation (S.T.), the Weill Neurohub Investigator Program (K.J.B., N.A.), an NSERC Predoctoral Fellowship (P.W.E.S.), the Ford Foundation Dissertation Fellowship (S.S.H.), the Weill Foundation Graduate Student Fellowship (S.S.H.), an NSF Graduate Fellowship (S.T.) and the NIH (grant no. R01 MH125978: K.J.B.; grant no. R01 MH136475: K.J.B., N.A.; grant nos. F32 MH125536 and K99 MH135209: A.D.N.; grant nos. R01 NS078118 and R01 NS121287: J.T.P.; grant no. R01 MH115045, R01 NS108874 and R01 MH118298: J.Q.P.; grant no. T32 GM007449: S.S.H.; grant no. MH126960: P.M.J.; grant no. R01 MH129751: S.J.S.), EMBO (ALTF 585-2021: C.A.), the Medical Research Council Centre of Research Excellence in Therapeutic Genomics (MR/Z504725/1: S.J.S., N.A.), the UCSF Bakar Aging Research Institute postdoctoral fellowship (C.A.) and the Bettencourt Schueller foundation (C.A.).

Author information

Author notes

  1. These authors contributed equally: Serena Tamura, Andrew D. Nelson, Perry W. E. Spratt

Authors and Affiliations

  1. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA

    Serena Tamura, Xujia Zhou, Zizheng Li, Coline Arnould, Vladyslav Barskyi, Beniamin Krupkin, Kiana Young, Jingjing Zhao, Navneet Matharu & Nadav Ahituv

  2. Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA

    Serena Tamura, Xujia Zhou, Zizheng Li, Coline Arnould, Vladyslav Barskyi, Beniamin Krupkin, Kiana Young, Jingjing Zhao, Navneet Matharu & Nadav Ahituv

  3. Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA

    Andrew D. Nelson, Perry W. E. Spratt, Elizabeth C. Hamada, Henry Kyoung, Atehsa Sahagun, Caroline M. Keeshen, Roy Ben-Shalom, Sunrae E. Taloma, Selin Schamiloglu, Ying C. Li, Stephan J. Sanders & Kevin J. Bender

  4. Department of Neurology, University of California San Francisco, San Francisco, CA, USA

    Andrew D. Nelson, Perry W. E. Spratt, Elizabeth C. Hamada, Henry Kyoung, Stephanie S. Holden, Atehsa Sahagun, Caroline M. Keeshen, Roy Ben-Shalom, Sunrae E. Taloma, Selin Schamiloglu, Ying C. Li, Jeanne T. Paz & Kevin J. Bender

  5. Gladstone Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA

    Stephanie S. Holden & Jeanne T. Paz

  6. Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA

    Congyi Lu & Jen Q. Pan

  7. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

    Lia Min & Paul M. Jenkins

  8. Department of Psychiatry, University of Michigan Medical School, Ann Arbor, MI, USA

    Paul M. Jenkins

  9. Department of Psychiatry, University of California San Francisco, San Francisco, CA, USA

    Stephan J. Sanders

  10. Institute of Developmental and Regenerative Medicine, Department of Paediatrics, University of Oxford, Oxford, UK

    Stephan J. Sanders

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Contributions

Conceptualization: K.J.B., S.J.S., N.A. Methodology: S.T., A.D.N., P.W.E.S., H.K., J.Z., N.M., J.Q.P., J.T.P., P.M.J., R.B.-S., N.A., K.J.B. Software: H.K., J.Z., R.B.-S. Formal analysis: S.T., A.D.N., P.W.E.S., V.B., B.K., L.M., P.M.J., H.K., J.Z., K.J.B. Investigation: S.T., A.D.N., P.W.E.S., E.C.H., X.Z., H.K., Z.L., C.A., V.B., K.Y., J.Z., S.S.H., A.S., C.M.K., C.L., S.E.T., S.S., Y.C.L., K.J.B. Writing—original draft: S.T., A.D.N., N.A., K.J.B. Writing—review and editing: all authors. Visualization: S.T., A.D.N., P.W.E.S., H.K., J.Z., K.J.B. Supervision: N.A., K.J.B. Funding acquisition: S.T., A.D.N., P.W.E.S., S.S.H., J.Q.P., J.T.P., S.J.S., N.A., K.J.B.

Corresponding authors

Correspondence to Nadav Ahituv or Kevin J. Bender.

Ethics declarations

Competing interests

N.M. is the cofounder and former board member and CSO of Regel Therapeutics, N.A. is the cofounder of Regel Therapeutics and both N.A. and K.J.B. are on the scientific advisory board of Regel Therapeutics. P.W.E.S. is a Program Director at Regel Therapeutics. N.M. and N.A. are the inventors on patent ‘Gene therapy for haploinsufficiency’ WO2018148256A9. N.A., K.J.B. and S.J.S. receive funding from BioMarin Pharmaceutical Incorporated. The other authors declare no competing interests.

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Nature thanks Elvir Becirovic, James Trimmer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Excitatory pyramidal neurons in the mPFC are GFP+ in Cre-negative Scn2a+/KI animals.

a. Coronal brain sections from P60 Scn2a+/KI mouse (Cre-) immunostained with anti-GFP and anti-parvalbumin (PV). b. Zoom of area highlighted by dashed box in panel a, with GFP and Parvalbumin channels separated then merged at bottom. Parv+ somata are circled in the GFP panel. c. Further zoom of region in layer 5b in panel b. Parv+ somata are circled as in panel c. d. Quantification of mean fluorescence intensify of GFP in PV-negative cells, PV-positive cells, and neuropil (area without somata as a measure of background fluorescence). Data are from 2 mice. Circles are mean GFP intensity values in ROIs of Parv- somata, Parv+ somata, or neighboring neuropil in single optical sections. Box plots are medians, quartiles, and 100% tails. n = 67 Parv- and 37 Parv+ cells analyzed; Parv- vs. Parv + : ****p < 0.0001. Parv- vs. neuropil: ****p < 0.0001. Holm-Šídák multiple comparisons test.

Source Data

Extended Data Fig. 2 In vitro optimization of CRISPRa constructs in mouse Neuroblastoma-2A cells.

a. Fold change of Scn2a expression in Neuro-2a cells transfected with plasmids containing sgRNAs targeting the promoter of mouse Scn2a compared to a no-sgRNA VP64 control. Blue bars represent plasmids with largest increase in Scn2a expression. Circles are replicates, overlaid on mean ± SD. b. Fold change of Scn2a transduced with rAAV-DJ virus in Neuro-2a cells. Circles are replicates, overlaid on mean ± SD. c. Total animal weight at time of 8 mg/kg 4-AP administration (animals aged 69–119 days). Circles are animals. Box plots are medians, quartiles and 100% tails. d. RT-qPCR analysis of dCas9 and mCherry mRNA within the mPFC of tail vein injected Scn2a+/+ + CRISPRa (light gray) and Scn2a+/− + CRISPRa (purple) versus uninjected controls Scn2a+/+ (dark gray) or Scn2a+/− (cyan). Injected animals with at least a 10-fold increase in expression levels of both dCas9 and mCherry to the average Scn2a+/+ uninjected controls were included in EEG datasets in Fig. 3.

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Extended Data Fig. 3 Scn2a-CRISPRa off-target analysis in cell lines.

a. RNA-seq expression levels of Scn2a TAD-domain genes from Scn2a-rAAV-CRISPRa treated Neuro-2a cells compared to VP64-only. Circles are individual replicates, box plots are medians, quartiles and 100% tails. p-values noted above data. Wald test. b. RNA-seq expression levels of sodium channels from Scn2a-rAAV-CRISPRa treated Neuro-2a cells compared to VP64-only. Circles are individual replicates, box plots are medians, quartiles and 100% tails. p-values noted above data. Wald test. c. Volcano plot representing Log2 fold-change in expression levels for each gene. Wald test. P-values noted above each comparison. Grey dots represent no significant DEGs for CRISPRa, purple dots signify Scn2a and nearby genes, and orange dots denote upregulated genes. d. Gene ontology (GO) analysis showing enrichment of molecular functions. e. Gene ontology (GO) analysis showing enrichment of biological processes. f. Analysis of sgRNA sequence off-targeting using Cas-OFFinder.

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Extended Data Fig. 4 Intrinsic electrophysiology of Scn2a+/KI and CRISPRa treated neurons.

a. AP threshold from P57-85 Scn2a-rAAV-empty vector Scn2a+/+ (light gray) and Scn2a+/− (magenta) neurons, Scn2a-rAAV-CRISPRa treated Scn2a+/+ (dark gray) and Scn2a+/− (purple) neurons, and Scn2a+/KI Cre- (green) and Scn2a+/KI Cre+ (gray) neurons. Threshold of the first AP evoked by a near-rheobase current. Scn2a+/+ + empty: n = 17 cells; Scn2a+/− + empty: n = 27 cells; Scn2a+/+ + CRISPRa: n = 24 cells; Scn2a+/− + CRISPRa: n = 19 cells; Scn2a+/KI Cre-: n = 29 cells; Scn2a+/KI Cre + : n = 21 cells. No significant differences. Holm-Šídák multiple comparisons test. Circles are individual cells, box plots are medians, quartiles and 100% tails. b. Input resistance (MΩ). Scn2a+/+ + empty: n = 17 cells; Scn2a+/− + empty: n = 27 cells; Scn2a+/+ + CRISPRa: n = 17 cells; Scn2a+/− + CRISPRa: n = 11 cells; Scn2a+/KI Cre-: n = 26 cells; Scn2a+/KI Cre + : n = 19 cells. No significant differences. Holm-Šídák multiple comparisons test. Circles are individual cells, box plots are medians, quartiles and 100% tails. c. Rheobase current (pA) to generate first spike. Scn2a+/+ + empty: n = 16 cells; Scn2a+/− + empty: n = 27 cells; Scn2a+/+ + CRISPRa: n = 17 cells; Scn2a+/− + CRISPRa: n = 12 cells; Scn2a+/KI Cre-: n = 25 cells; Scn2a+/KI Cre + : n = 19 cells. No significant differences. Holm-Šídák multiple comparisons test. Circles are individual cells, box plots are medians, quartiles and 100% tails. d. APs per 300 ms stimulation epoch for each current amplitude. Left: Quantification of firing rate slope of data on Right. Scn2a+/+ + empty: n = 16 cells; Scn2a+/− + empty: n = 27 cells; Scn2a+/+ + CRISPRa: n = 17 cells; Scn2a+/− + CRISPRa: n = 12 cells; Scn2a+/KI Cre-: n = 25 cells; Scn2a+/KI Cre + : n = 19 cells. No significant differences. Holm-Šídák multiple comparisons test. Circles are individual cells, box plots are medians, quartiles and 100% tails.Right: Number of APs versus current amplitude injected. Circles and bars are mean ± SEM at each stimulus intensity.

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Extended Data Fig. 5 Scn2a-CRISPRa off-target analysis in the mouse neocortex.

a. RNA-seq expression levels of Scn2a TAD-domain genes from Scn2a-rAAV-CRISPRa treated Neuro-2a cells compared to VP64-only. Circles are individual mice, box plots are medians, quartiles and 100% tails. b. RNA-seq expression levels of sodium channels from Scn2a-rAAV-CRISPRa treated Neuro-2a cells compared to VP64-only. Circles are individual mice, box plots are medians, quartiles and 100% tails. c. Volcano plot representing Log2 fold-change in expression levels for each gene for WT vs Scn2a+/− “Het”. Wald test. P-values noted above each comparison. Grey dots represent no significant DEGs for CRISPRa, purple dots signify Scn2a and nearby genes, and orange dots denote upregulated genes. d. Same as c, but for CRISPRa-treated Scn2a+/− “CRISPRa” vs WT. e. Same as c, but for CRISPRa-treated Scn2a+/− “CRISPRa” vs Scn2a+/− “Het”. f. Analysis of sgRNA sequence off-targeting using Cas-OFFinder. g. Data representation of NeuN-positive neuronal nuclei isolated from cortical tissue for FACS sorting. Representative FACS plots visualized with FlowJo V10 with percentage of parent gates for each population. Singlets were selected based on FSC-H versus FSC-A. h. From singlets, DAPI-positive events were gated to identify nuclei. i. NeuN-positive neuronal nuclei were selected using an anti-NeuN antibody conjugated to Alexa Fluor 488.

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Extended Data Fig. 6 CRISPRa expression persists through 16 months post-systemic injection.

Peak AP dV/dt (top) at 30-, 56-, 107-, and 210-days following tail-vein injection of Scn2a-rAAV-CRISPRa-PHP.eB in Scn2a+/−mice (purple). Circles represent individual neurons. Lines are linear regression. qPCR of mCherry (middle) or dCas9 (bottom) mRNA from neocortical samples collected from tail-vein or retro-orbital Scn2a-rAAV-CRISPRa-PHP.eB injected Scn2a+/+ (gray) or Scn2a+/− mice (purple) across time. Circles represent single mice. Lines are linear regression.

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Extended Data Fig. 7 Behavioral and EEG response to increasing doses of 4-AP in Scn2a+/− mice.

a. Example PFC EEGs from animals receiving 8 and 15 mg/kg 4-AP. 4-AP administered at onset of all recordings. Dashed box denotes a tonic clonic seizure and subsequent mortality occurring with 15 mg/kg dosing in Scn2a+/− animal. EEG data within boxed area is shown at higher resolution on right. b. Survival curves for all WT and Scn2a+/− animals for 6, 8 and 15 mg/kg 4-AP. WT: black, Scn2a+/−: cyan.

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Extended Data Fig. 8 In vitro optimization of CRISPRa constructs in human SH-SY5Ycells.

a. Fold change of SCN2A expression in SH-SY5Y cells transfected with plasmids containing sgRNAs targeting the promoter of human SCN2A compared to a no sgRNA VP64 control. b. Fold change of SCN2A transduced with rAAV-DJ virus in human SH-SY5Y cells. c. SCN2A mRNA expression from SCN2A+/+ (black), SCN2A+/− (cyan), and SCN2A-rAAV-CRISPRa treated SCN2A+/− (purple) hESC-derived neurons normalized to wild type average. SCN2A+/+ vs. SCN2A+/−: *p = 0.03. Holm-Šídák multiple comparisons test. n = 3 replicates for all conditions. circles are individual replicates. Bars are mean ± SD.

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Extended Data Fig. 9 Axon initial segment structural plasticity occurs in SCN2A+/− neurons and is rescued by CRISPRa.

a. Representative images of SCN2A+/+ (black) and SCN2A+/− (cyan) human stem-cell-derived neurons immunostained with antibodies against ankyrin-G (green) and MAP2 (magenta). Arrowheads denote start and end points used to quantify AIS length. b. Quantification of AIS length. SCN2A+/+: n = 56 cells, 3 dishes. SCN2A+/−: n = 56 cells, 3 dishes. ****p < 0.0001. Mann-Whitney test. c. Representative images of SCN2A+/− neurons expressing Scn2a-rAAV-CRISPRa-mCherry (purple) and mCherry-negative internal SCN2A+/− controls (cyan). Immunostaining against ankyrin-G and MAP2. d. Quantification of AIS length. SCN2A+/−: n = 122 cells, 3 dishes. SCN2A+/− + CRISPRa: n = 23 cells, 3 dishes. ****p < 0.0001. Mann-Whitney test. Circles are individual AIS calculations. Box plots are medians, quartiles, and 100% tails.

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Tamura, S., Nelson, A.D., Spratt, P.W.E. et al. CRISPR activation for SCN2A-related neurodevelopmental disorders. Nature 646, 983–991 (2025). https://doi.org/10.1038/s41586-025-09522-w

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