Nataliya Basalova, Maria Illarionova, Mariya Skryabina, Maksim Vigovskiy, Anastasia Tolstoluzhinskaya, Alexandra Primak, Elizaveta Chechekhina, Vadim Chechekhin, Maxim Karagyaur, Anastasia Efimenko
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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 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.
Non-Coding RNABiochemistry, Genetics and Molecular Biology-Genetics
CiteScore
6.70
自引率
4.70%
发文量
74
审稿时长
10 weeks
期刊介绍:
Functional studies dealing with identification, structure-function relationships or biological activity of: small regulatory RNAs (miRNAs, siRNAs and piRNAs) associated with the RNA interference pathway small nuclear RNAs, small nucleolar and tRNAs derived small RNAs other types of small RNAs, such as those associated with splice junctions and transcription start sites long non-coding RNAs, including antisense RNAs, long ''intergenic'' RNAs, intronic RNAs and ''enhancer'' RNAs other classes of RNAs such as vault RNAs, scaRNAs, circular RNAs, 7SL RNAs, telomeric and centromeric RNAs regulatory functions of mRNAs and UTR-derived RNAs catalytic and allosteric (riboswitch) RNAs viral, transposon and repeat-derived RNAs bacterial regulatory RNAs, including CRISPR RNAS Analysis of RNA processing, RNA binding proteins, RNA signaling and RNA interaction pathways: DICER AGO, PIWI and PIWI-like proteins other classes of RNA binding and RNA transport proteins RNA interactions with chromatin-modifying complexes RNA interactions with DNA and other RNAs the role of RNA in the formation and function of specialized subnuclear organelles and other aspects of cell biology intercellular and intergenerational RNA signaling RNA processing structure-function relationships in RNA complexes RNA analyses, informatics, tools and technologies: transcriptomic analyses and technologies development of tools and technologies for RNA biology and therapeutics Translational studies involving long and short non-coding RNAs: identification of biomarkers development of new therapies involving microRNAs and other ncRNAs clinical studies involving microRNAs and other ncRNAs.