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

IF 3.6 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Non-Coding RNA Pub Date : 2023-08-24 DOI:10.3390/ncrna9050049

Nataliya Basalova, Maria Illarionova, Mariya Skryabina, Maksim Vigovskiy, Anastasia Tolstoluzhinskaya, Alexandra Primak, Elizaveta Chechekhina, Vadim Chechekhin, Maxim Karagyaur, Anastasia Efimenko

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引用次数: 0

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.

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CRISPR/Cas9技术用于人间充质干细胞非编码RNA基因敲除的进展和障碍。

近年来，非编码RNA（ncRNAs）基因因其广泛参与生理和病理过程以及调控网络而引起越来越多的关注。对ncRNA功能和分子伴侣的研究为早期诊断和治疗以前无法治愈的疾病开辟了机会。然而，由于一些特定的问题，ncRNA功能分析中的经典“功能损失”方法受到了挑战。在这里，我们研究了两种CRISPR/Cas9变体，野生型（SpCas9wt）和镍酶（SpCas9 D10A）可编程核酸酶，用于编辑人间充质基质细胞（MSC）中的扩展DNA序列的效力。编辑纤维化相关的hsa-miR-21-5p和hsa-miR-29c-3p的基因，我们已经表明一对SpCas9D10A分子可以有效地破坏MSC基因组中的miRNA基因。这不仅导致MSC和MSC产生的细胞外小泡中敲除miRNA的水平降低，还导致细胞生理学和细胞分泌组抗纤维化特性的改变。这些变化与先前发表的敲低某些miRNA的数据有很好的相关性。所提出的方法可用于敲除MSC或类似细胞类型基因组中的ncRNA基因，以研究其在生物过程中的功能。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Non-Coding RNA Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

6.70

自引率

4.70%

发文量

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.