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在使用CRISPR/Cas9技术的进步和障碍,用于在人间半机体干细胞中进行非编码RNA基因淘汰

Nataliya Basalova^1,2, Maria Illarionova², Mariya Skryabina²

¹Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 27/10, Lomonosovsky Ave., 119192 Moscow, Russia.

|September 22, 2023

概括

克里斯普尔/卡斯9尼克酶有效地破坏人类介质细胞 (MSCs) 中的非编码RNA基因. 这种基因编辑方法改变了细胞生理和机密性质,为研究ncRNA功能和疾病治疗提供了新的途径.

科学领域:

分子生物学分子生物学
基因编辑基因编辑
细胞生物学细胞生物学

背景情况:

非编码RNAs (ncRNAs) 在生理和病理过程中起着至关重要的作用.
研究ncRNA功能对于开发疾病的诊断和治疗至关重要.
对于ncRNA分析的经典功能丧失方法面临挑战.

研究的目的:

评估CRISPR/Cas9变体 (SpCas9wt和SpCas9D10A) 对于编辑人类介质细胞 (MSCs) 中扩展DNA序列的疗效.
使用CRISPR/Cas9技术研究特定的microRNA (miRNA) 基因 (hsa-miR-21-5p和hsa-miR-29c-3p) 的破坏.
评估ncRNA基因干扰对MSC功能和机密性质的影响.

主要方法:

使用了两个CRISPR/Cas9变体:野生类型 (SpCas9wt) 和尼克酶 (SpCas9D10A).
在人类MSC中针对和编辑纤维化相关的miRNA基因 (hsa-miR-21-5p和hsa-miR-29c-3p).
在MSC及其细胞外囊中的量化miRNA水平后编辑.
分析了细胞生理学的变化和MSC分泌物的抗纤维素特性.

主要成果:

一对SpCas9D10A分子有效地破坏了MSC基因组中的目标miRNA基因.
在MSC及其分泌的细胞外囊中观察到miRNAs的成功淘汰.
破坏miRNA基因导致MSC细胞生理学的显著改变.
该研究观察到MSC分泌物的抗纤维素特性发生变化,与已知的miRNA淘汰效应相关.

结论:

克里斯普尔/卡斯9尼克酶是破坏MSCs中ncRNA基因的有力工具.
这种方法可以通过改变细胞和秘密特征来研究ncRNA的功能性.
开发的方法提供了一个有价值的策略,用于调查ncRNA在MSCs内的生物过程和疾病中的作用.

关键词:

这就是CRISPR/Cas9的作用.在SpCas9D10A中使用.有纤维化的纤维化.基因组编辑基因组编辑在hsa-miR-21中使用.这是一个hsa-miR-29cc.介质细胞层细胞.这是一个小RNARNA.这是一个ncRNARNA.一些秘密一些秘密.

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