Skip to main content

Thank you for visiting nature.com. 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.

  • Correspondence
  • Published:

Prime editing-based gene correction alleviates the hyperexcitable phenotype and seizures of a genetic epilepsy mouse model

Acta Pharmacologica Sinica volume 44, pages 2342–2345 (2023)Cite this article

Dear Editor,

We derived mouse embryonic stem cells (mESCs) bearing the Kcnq2R207W mutation from corresponding mouse embryos. First, we applied an adenine base editor (ABE) to correct the mutated T nucleotide to C at the target site in the Kcnq2R207W mESCs (Fig. 1b). With no NGG PAM adjacent to the target site, we used NG-ABEmax [15] to introduce the specific transition (Figs. 1b and S1a). However, NG-ABEmax showed very low (0.45% ± 0.08%) editing efficiency (Fig. 1b). We next examined the homologous recombination (HR)-based method [16] to correct the Kcnq2R207W mutation using an HR donor with 800 bp or 500 bp homology arms (Figs. 1b and S1b). Although HR showed ~45% T to C editing efficiency, the indel frequency was as high as 56.21% (Fig. 1b), which may indicate safety concerns in clinical application. The PE system was then used to correct point mutations in the Kcnq2R207W mESCs (Fig. S2a). To find the best prime editing guide RNA (pegRNA) and nicking guide RNA (ngRNA) targeting the Kcnq2R207W mutation, we designed 7 pegRNAs and tested their editing outcome in mESCs (Table S1). The results showed that pegRNA4 exhibited the highest editing efficiency (12.12% ± 1.15%) (Figs. 1c and S2a). We then screened 7 ngRNAs together with each of the 7 pegRNAs for their performance in Kcnq2R207W mESCs (Figs. 1d and S2a, c and Table S1). By deep sequencing analysis of the frequency with specified edits or indels, we found that the combination of pegRNA4 and ngRNA15 achieved the highest editing efficiency (41.37% ± 1.43%) and markedly reduced the level of indels compared with HR (14.34% vs. 36.68%) (Figs. 1b–d and S2a, c). In addition, this combination showed rare off-target effects on sites with sequences similar to pegRNA4 and ngRNA15 (Figs. 1e and S3a–c and Table S2). Moreover, the PE3 system was transduced into primary cortical neurons from E18.5 Kcnq2R207W homozygote mice through lentiviral delivery (Fig. S2b). The neurons were collected 2 weeks after transduction, and successfully transduced neurons were sorted through flow cytometry. We observed an average of 14.61% editing with 2.86% indels in unsorted neurons and 43.16% editing with 2.55% indels in sorted neurons (Fig. 1f). These data indicate that PE3 can efficiently correct the Kcnq2R207W mutation in mESCs and neurons.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Efficient on-target editing of mutations by a prime editor alleviates the hyperexcitable phenotype and seizures of the Kcnq2R207W mouse model.

References

  1. Ali A. Global health: epilepsy. Semin Neurol. 2018;38:191–9.

    Article  PubMed  Google Scholar 

  2. Carcak N, Ozkara C. Seizures and antiepileptic drugs: from pathophysiology to clinical practice. Curr Pharm Des. 2017;23:6376–88.

    Article  CAS  PubMed  Google Scholar 

  3. Eskioglou E, Perrenoud MP, Ryvlin P, Novy J. Novel treatment and new drugs in epilepsy treatment. Curr Pharm Des. 2017;23:6389–98.

    Article  CAS  PubMed  Google Scholar 

  4. Loscher W, Potschka H, Sisodiya SM, Vezzani A. Drug resistance in epilepsy: clinical impact, potential mechanisms, and new innovative treatment options. Pharmacol Rev. 2020;72:606–38.

    Article  PubMed  PubMed Central  Google Scholar 

  5. McTague A, Howell KB, Cross JH, Kurian MA, Scheffer IE. The genetic landscape of the epileptic encephalopathies of infancy and childhood. Lancet Neurol. 2016;15:304–16.

    Article  PubMed  Google Scholar 

  6. Specchio N, Curatolo P. Developmental and epileptic encephalopathies: what we do and do not know. Brain. 2021;144:32–43.

    Article  PubMed  Google Scholar 

  7. Abraham AA, Tisdale JF. Gene therapy for sickle cell disease: moving from the bench to the bedside. Blood. 2021;138:932–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Leebeek FWG, Miesbach W. Gene therapy for hemophilia: a review on clinical benefit, limitations, and remaining issues. Blood. 2021;138:923–31.

    Article  CAS  PubMed  Google Scholar 

  9. Lin FL, Wang PY, Chuang YF, Wang JH, Wong VHY, Bui BV, et al. Gene therapy intervention in neovascular eye disease: a recent update. Mol Ther. 2020;28:2120–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Apte RS. Gene therapy for retinal degeneration. Cell. 2018;173:5.

    Article  CAS  PubMed  Google Scholar 

  11. Colasante G, Qiu Y, Massimino L, Di Berardino C, Cornford JH, Snowball A, et al. In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy. Brain. 2020;143:891–905.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Colasante G, Lignani G, Brusco S, Di Berardino C, Carpenter J, Giannelli S, et al. dCas9-based Scn1a gene activation restores inhibitory interneuron excitability and attenuates seizures in Dravet syndrome mice. Mol Ther. 2020;28:235–53.

    Article  CAS  PubMed  Google Scholar 

  13. Tian F, Cao B, Xu H, Zhan L, Nan F, Li N, et al. Epilepsy phenotype and response to KCNQ openers in mice harboring the Kcnq2 R207W voltage-sensor mutation. Neurobiol Dis. 2022;174:105860.

    Article  CAS  PubMed  Google Scholar 

  14. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Huang TP, Zhao KT, Miller SM, Gaudelli NM, Oakes BL, Fellmann C, et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat Biotechnol. 2019;37:626–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 2017;27:801–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Blumkin L, Suls A, Deconinck T, De Jonghe P, Linder I, Kivity S, et al. Neonatal seizures associated with a severe neonatal myoclonus like dyskinesia due to a familial KCNQ2 gene mutation. Eur J Paediatr Neurol. 2012;16:356–60.

    Article  PubMed  Google Scholar 

  18. Dedek K, Kunath B, Kananura C, Reuner U, Jentsch TJ, Steinlein OK. Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K+ channel. Proc Natl Acad Sci USA. 2001;98:12272–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu Y, Yang G, Huang S, Li X, Wang X, Li G, et al. Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Res. 2021;31:1134–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015;33:985–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Finn JD, Smith AR, Patel MC, Shaw L, Youniss MR, van Heteren J, et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 2018;22:2227–35.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Hui Yang for his insightful comments on this study. We thank Pei Wang, Yong-jie Cai, Jun-jie Tang, De-lun Kong for help in experiment on this study. This study was supported by the High-level new R&D institute (2019B090904008) and the High-level Innovative Research Institute (2021B0909050003) of the Department of Science and Technology of Guangdong Province, the National Science Fund for Distinguished Young Scholars (81825021), and National Science and Technology Innovation 2030 Major Program (2021ZD0200900). The diagrams were created with Biorender.com.

Author information

Author notes

  1. These authors contributed equally: Bi-rong Cao, Yi-ming Huang

Authors and Affiliations

  1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

    Bi-rong Cao, Fu-yun Tian, Qian-bei Guo, Hai-yan Xu, Li Zhan & Zhao-bing Gao

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Bi-rong Cao & Zhao-bing Gao

  3. Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, 200031, China

    Yi-ming Huang, Jin-hui Li, Chun-long Xu, Rui-min Lv, Yi-di Sun, Xin-de Hu & Chang-yang Zhou

  4. Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China

    Fu-yun Tian & Zhao-bing Gao

  5. Shanghai Immunocan Biotech Co., LTD, Shanghai, 201800, China

    Yu Wei & Ji-kai Liu

Authors
  1. Bi-rong Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi-ming Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fu-yun Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jin-hui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chun-long Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yu Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ji-kai Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Qian-bei Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hai-yan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Li Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Rui-min Lv
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yi-di Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Xin-de Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Zhao-bing Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Chang-yang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

BRC and FYT designed and performed experiments. YMH and YDS performed data analysis. JHL, CLX and RML performed PCR analysis and FACS. YW and JKL performed the zygote injection. QBG, HYX and LZ performed the open field test, 6-HZ and PTZ test. BRC, FYT, ZBG, YDS and CYZ wrote the paper. YDS, XDH, ZBG and CYZ supervised the project.

Corresponding authors

Correspondence to Yi-di Sun, Xin-de Hu, Zhao-bing Gao or Chang-yang Zhou.

Ethics declarations

Competing interests

Yu Wei and Ji-kai Liu are employees of the Shanghai Immunocan Biotech Co., LTD. The other authors disclose no competing interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cao, Br., Huang, Ym., Tian, Fy. et al. Prime editing-based gene correction alleviates the hyperexcitable phenotype and seizures of a genetic epilepsy mouse model. Acta Pharmacol Sin 44, 2342–2345 (2023). https://doi.org/10.1038/s41401-023-01115-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41401-023-01115-5

Search

Advanced search

Quick links