Basalova Nataliya, Illarionova Maria, Skryabina Mariya, Vigovskiy Maksim, Tolstoluzhinskaya Anastasia, Primak Alexandra, Chechekhina Elizaveta, Chechekhin Vadim, Karagyaur Maxim, Efimenko Anastasia
Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 27/10, Lomonosovsky Ave., 119192 Moscow, Russia.
Faculty of Medicine, Lomonosov Moscow State University, 27/1, Lomonosovsky Ave., 119192 Moscow, Russia.
Noncoding RNA. 2023 Aug 24;9(5):49. doi: 10.3390/ncrna9050049.
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.
近年来，非编码RNA（ncRNAs）基因因其广泛参与生理和病理过程及调控网络而受到越来越多的关注。对ncRNAs功能及其分子伴侣的研究为以前无法治愈的疾病的早期诊断和治疗带来了机遇。然而，由于一些特定问题，ncRNA功能分析中的经典“功能丧失”方法受到了挑战。在此，我们研究了两种CRISPR/Cas9变体，即野生型（SpCas9wt）和切口酶（SpCas9D10A）可编程核酸酶，对人间充质基质细胞（MSCs）中延伸DNA序列进行编辑的效能。通过编辑与纤维化相关的hsa-miR-21-5p和hsa-miR-29c-3p基因，我们发现一对SpCas9D10A分子能够有效破坏MSCs基因组内的miRNA基因。这不仅导致MSCs及MSCs产生的细胞外囊泡中敲除miRNA水平降低，还引起细胞生理学变化以及细胞分泌组的抗纤维化特性改变。这些变化与先前发表的某些miRNA敲低数据高度相关。所提出的方法可用于敲除MSCs或类似细胞类型基因组内的ncRNA基因，以研究它们在生物过程中的功能。
