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

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 tre...

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Bibliographic Details
Published in:Non-coding RNA 2023-08, Vol.9 (5), p.49
Main Authors: Basalova, Nataliya, Illarionova, Maria, Skryabina, Mariya, Vigovskiy, Maksim, Tolstoluzhinskaya, Anastasia, Primak, Alexandra, Chechekhina, Elizaveta, Chechekhin, Vadim, Karagyaur, Maxim, Efimenko, Anastasia
Format: Article
Language:English
Subjects:
Analysis
Chromosome 5
Cloning
CRISPR
CRISPR/Cas9
DNA binding proteins
DNA repair
DNA sequencing
Efficiency
Fibrosis
Genes
genome editing
Genomes
Genomics
hsa-miR-21
hsa-miR-29c
Mesenchymal stem cells
MicroRNA
MicroRNAs
miRNA
ncRNA
Non-coding RNA
Nuclease
Nucleases
Nucleotide sequence
Nucleotide sequencing
Secretome
Stem cells
Stromal cells
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Summary: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.
ISSN:2311-553X
2311-553X
DOI:10.3390/ncrna9050049