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通过结合CRISPRa介导的内源性Gata4激活和外源Mef2c和Tbx5表达的直接心脏重编程

Peisen Huang^1,2, Jun Xu^1,2, Benjamin Keepers^1,2

¹McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Molecular therapy. Nucleic acids

|December 25, 2024

概括

科学家使用CRISPR激活来促进内源基因表达,通过激活GATA4成功将纤维细胞转化为诱导心肌细胞 (iCMs). 这减少了对心脏再生的病毒载体的依赖.

科学领域:

心血管生物学心血管生物学
基因编辑技术的技术
再生医学是一种再生医学.

背景情况:

直接的心脏重编程通过转录因子 (TF) 将纤维细胞转化为诱导心肌细胞 (iCMs).
TFs的病毒载体传递带有基因组突变和免疫反应等风险,限制了临床使用.
激活内源性TF提供了一个比外源性TF交付更安全的替代方案.

研究的目的:

为了研究CRISPR激活 (CRISPRa) 对于交换激活内源心脏重编程因子 (MEF2C,GATA4,TBX5).
评估CRISPRa在诱导来自小鼠和人类纤维细胞的iCMs中的有效性.
探索一种新的心脏再生策略,减少对外部因素的依赖.

主要方法:

使用修改后的CRISPRa/dCas9系统来向内源MEF2C,GATA4和TBX5.5的促进剂和增强剂.
选单导向RNAs (sgRNAs),以确定那些能够激活TF基因表达的人.
结合CRISPRa介导的GATA4激活与外源MEF2C和TBX5表达.

主要成果:

确定了有效激活内源GATA4表达的sgRNAs.
通过使用CRISPRa激活的GATA4以及外源MEF2C和TBX5.5成功地将纤维细胞转化为iCMs.
观察到对内源MEF2C和TBX5的CRISPRa介导的交换活化效果有限,可能是由于表观遗传障碍.

结论:

通过CRISPRa介导的内源GATA4的激活,与外源MEF2C和TBX5相结合,为减少对心脏重编程中的外源因素的依赖提供了概念证明.
这种方法提供了一个新的战略,用于将痕形成纤维细胞转化为iCMs用于再生医学.
需要进一步的技术改进,才能实现仅通过CRISPR (所有内源因素的激活) 重编程.

关键词:

这就是CRISPRdCas9系统.许多MGTGT的产品.MT:RNA/DNA编辑编辑通过直接的心脏重编程.内源性交易的激活.导向RNA指导RNA是指导RNA的指导RNA.

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