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Biochemistry (Moscow)

, Volume 84, Issue 9, pp 1074–1084 | Cite as

Methods for Correction of the Single-Nucleotide Substitution c.840C>T in Exon 7 of the SMN2 Gene

  • K. R. ValetdinovaEmail author
  • V. S. Ovechkina
  • S. M. Zakian
Article
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Abstract

The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients’ genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.

Keywords

spinal muscular atrophy induced pluripotent stem cells motor neuron progenitors gene editing 

Abbreviations

iPSCs

induced pluripotent stem cells

MNP

motor neuron progenitor

SMA

spinal muscular atrophy

sgRNA

single guide RNA

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Notes

Acknowledgements

The authors are grateful to A. A. Nemudryi for help in assembling pX552_miCMV_Puro_SMN.

Funding. This work was supported by the Russian Science Foundation (project 17-75-10041).

Ethical approval. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

References

  1. 1.
    Verhaart, I. E., Robertson, A., Leary, R., McMacken, G., Konig, K., Kirschner, J., Jones, C. C., Cook, S. F., and Lochmuller, H. (2017) A multi-source approach to determine SMA incidence and research ready population, J. Neurol., 264, 1465–1473, doi:  https://doi.org/10.1007/s00415-017-8549-1.CrossRefGoogle Scholar
  2. 2.
    Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet, L., Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M., Le Paslier, D., Frezal, J., Cohen, D., Weissenbach, J., Munnich, A., and Melki, J. (1995) Identification and characterization of a spinal muscular atrophy-determining gene, Cell, 80, 155–165, doi: https://doi.org/0092-8674(95)90460-3.CrossRefGoogle Scholar
  3. 3.
    Monani, U. R., Lorson, C. L., Parsons, D. W., Prior, T. W., Androphy, E. J., Burghes, A. H., and McPherson, J. D. (1999) A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2, Hum. Mol. Genet., 8, 1177–1183.CrossRefGoogle Scholar
  4. 4.
    Cartegni, L., and Krainer, A. R. (2002) Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1, Nat. Genet., 30, 377–384, doi:  https://doi.org/10.1038/ng854.CrossRefGoogle Scholar
  5. 5.
    Kashima, T., and Manley, J. L. (2003) A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy, Nat. Genet., 34, 460–463, doi:  https://doi.org/10.1038/ng1207.CrossRefGoogle Scholar
  6. 6.
    McAndrew, P. E., Parsons, D. W., Simard, L. R., Rochette, C., Ray, P. N., Mendell, J. R., Prior, T. W., and Burghes, A. H. (1997) Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number, Am. J. Hum. Genet., 60, 1411–1422, doi:  https://doi.org/10.1086/515465.CrossRefGoogle Scholar
  7. 7.
    Gidaro, T., and Servais, L. (2019) Nusinersen treatment of spinal muscular atrophy: current knowledge and existing gaps, Dev. Med. Child Neurol., 61, 19–24, doi:  https://doi.org/10.1111/dmcn.14027.CrossRefGoogle Scholar
  8. 8.
    Liang, X., Potter, J., Kumar, S., Ravinder, N., and Chesnut, J. D. (2017) Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA, J. Biotechnol., 241, 136–146, doi:  https://doi.org/10.1016/j.jbiotec.2016.11.011.CrossRefGoogle Scholar
  9. 9.
    Valetdinova, K. R., Maretina, M. A., Kuranova, M. L., Grigor’eva, E. V., Minina, Y. M., Kizilova, E. A., Kiselev, A. V., Medvedev, S. P., Baranov, V. S., and Zakian, S. M. (2019) Generation of two spinal muscular atrophy (SMA) type I patient-derived induced pluripotent stem cell (iPSC) lines and two SMA type II patient-derived iPSC lines, Stem Cell Res., 34, 101376, doi:  https://doi.org/10.1016/j.scr.2018.101376.CrossRefGoogle Scholar
  10. 10.
    Du, Z. W., Chen, H., Liu, H., Lu, J., Qian, K., Huang, C. L., Zhong, X., Fan, F., and Zhang, S. C. (2015) Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells, Nat. Commun., 6, 6626, doi:  https://doi.org/10.1038/ncomms7626.CrossRefGoogle Scholar
  11. 11.
    Clarke, R., Heler, R., MacDougall, M. S., Yeo, N. C., Chavez, A., Regan, M., Hanakahi, L., Church, G. M., Marraffini, L. A., and Merrill, B. (2018) Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double strand DNA breaks, Mol. Cell, 71, 42–55, doi:  https://doi.org/10.1016/j.molcel.2018.06.005.CrossRefGoogle Scholar
  12. 12.
    Brinkman, E. K., Chen, T., Amendola, M., and van Steensel, B. (2014) Easy quantitative assessment of genome editing by sequence trace decomposition, Nucleic Acids Res., 42, e168, doi:  https://doi.org/10.1093/nar/gku936.CrossRefGoogle Scholar
  13. 13.
    Ishida, K., Gee, P., and Hotta, A. (2015) Minimizing off-target mutagenesis risks caused by programmable nucleases, Int. J. Mol. Sci., 16, 24751–24771, doi:  https://doi.org/10.3390/ijms161024751.CrossRefGoogle Scholar
  14. 14.
    Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V., Sharp, P. A., and Zhang, F. (2015) In vivo genome editing using Staphylococcus aureus Cas9, Nature, 520, 186–191, doi:  https://doi.org/10.1038/nature14299.CrossRefGoogle Scholar
  15. 15.
    Lin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., Wile, B. M., Vertino, P. M., Stewart, F. J., and Bao, G. (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences, Nucleic Acids Res., 42, 7473–7485, doi:  https://doi.org/10.1093/nar/gku402.CrossRefGoogle Scholar
  16. 16.
    Nemudryi, A. A., Valetdinova, K. R., Medvedev, S. P., and Zakian, S. M. (2014) TALEN and CRISPR/Cas genome editing systems: tools of discovery, Acta Naturae, 6, 19–40.CrossRefGoogle Scholar
  17. 17.
    Song, F., and Stieger, K. (2017) Optimizing the DNA donor template for homology-directed repair of double-strand breaks, Mol. Ther. Nucleic Acids, 7, 53–60, doi:  https://doi.org/10.1016/j.omtn.2017.02.006.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • K. R. Valetdinova
    • 1
    • 2
    • 3
    • 4
    Email author
  • V. S. Ovechkina
    • 1
    • 2
    • 3
    • 4
  • S. M. Zakian
    • 1
    • 2
    • 3
    • 4
  1. 1.Federal Research Center Institute of Cytology and GeneticsSiberian Branch of the Russian Academy of SciencesNovosibirskRussia
  2. 2.Institute of Chemical Biology and Fundamental MedicineSiberian Branch of the Russian Academy of SciencesNovosibirskRussia
  3. 3.Meshalkin National Medical Research CentreMinistry of Healthcare of Russian FederationNovosibirskRussia
  4. 4.Novosibirsk State UniversityNovosibirskRussia

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