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Article

Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells

by
Nataliya Basalova
1,2,
Maria Illarionova
2,
Mariya Skryabina
2,
Maksim Vigovskiy
1,2,
Anastasia Tolstoluzhinskaya
1,2,
Alexandra Primak
2,
Elizaveta Chechekhina
2,
Vadim Chechekhin
2,
Maxim Karagyaur
1,2,*,† and
Anastasia Efimenko
1,2,*,†
1
Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 27/10, Lomonosovsky Ave., 119192 Moscow, Russia
2
Faculty of Medicine, Lomonosov Moscow State University, 27/1, Lomonosovsky Ave., 119192 Moscow, Russia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Non-Coding RNA 2023, 9(5), 49; https://doi.org/10.3390/ncrna9050049
Submission received: 3 July 2023 / Revised: 17 August 2023 / Accepted: 22 August 2023 / Published: 24 August 2023
(This article belongs to the Section Evolution of Non-Coding RNA)
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Abstract

Non-coding RNA (ncRNAs) genes have attracted increasing attention in recent years due to their widespread involvement in physiological and pathological processes and regulatory networks. The study of the function and molecular partners of ncRNAs opens up opportunities for the early diagnosis and treatment of previously incurable diseases. However, the classical “loss-of-function” approach in ncRNA function analysis is challenged due to some specific issues. Here, we have studied the potency of two CRISPR/Cas9 variants, wild-type (SpCas9wt) and nickase (SpCas9D10A) programmable nucleases, for the editing of extended DNA sequences in human mesenchymal stromal cells (MSCs). Editing the genes of fibrosis-related hsa-miR-21-5p and hsa-miR-29c-3p, we have shown that a pair of SpCas9D10A molecules can effectively disrupt miRNA genes within the genomes of MSCs. This leads not only to a decrease in the level of knockout miRNA in MSCs and MSC-produced extracellular vesicles, but also to a change in cell physiology and the antifibrotic properties of the cell secretome. These changes correlate well with previously published data for the knockdown of certain miRNAs. The proposed approach can be used to knock out ncRNA genes within the genomes of MSCs or similar cell types in order to study their function in biological processes.
Keywords: ncRNA; miRNA; hsa-miR-21; hsa-miR-29c; genome editing; CRISPR/Cas9; SpCas9D10A; mesenchymal stromal cells; secretome; fibrosis ncRNA; miRNA; hsa-miR-21; hsa-miR-29c; genome editing; CRISPR/Cas9; SpCas9D10A; mesenchymal stromal cells; secretome; fibrosis

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MDPI and ACS Style

Basalova, N.; Illarionova, M.; Skryabina, M.; Vigovskiy, M.; Tolstoluzhinskaya, A.; Primak, A.; Chechekhina, E.; Chechekhin, V.; Karagyaur, M.; Efimenko, A. Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells. Non-Coding RNA 2023, 9, 49. https://doi.org/10.3390/ncrna9050049

AMA Style

Basalova N, Illarionova M, Skryabina M, Vigovskiy M, Tolstoluzhinskaya A, Primak A, Chechekhina E, Chechekhin V, Karagyaur M, Efimenko A. Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells. Non-Coding RNA. 2023; 9(5):49. https://doi.org/10.3390/ncrna9050049

Chicago/Turabian Style

Basalova, Nataliya, Maria Illarionova, Mariya Skryabina, Maksim Vigovskiy, Anastasia Tolstoluzhinskaya, Alexandra Primak, Elizaveta Chechekhina, Vadim Chechekhin, Maxim Karagyaur, and Anastasia Efimenko. 2023. "Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells" Non-Coding RNA 9, no. 5: 49. https://doi.org/10.3390/ncrna9050049

APA Style

Basalova, N., Illarionova, M., Skryabina, M., Vigovskiy, M., Tolstoluzhinskaya, A., Primak, A., Chechekhina, E., Chechekhin, V., Karagyaur, M., & Efimenko, A. (2023). Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells. Non-Coding RNA, 9(5), 49. https://doi.org/10.3390/ncrna9050049

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