紧的工程人类机械敏感的交换激活模块使强大的和多功能合成转录控制
Barun Mahata1, Alan Cabrera1, Daniel A Brenner1
1Department of Bioengineering, Rice University, Houston, TX, USA.
Nature methods
|October 9, 2023
概括
科学家们从人类机械敏感因子中设计出紧而强大的转录激活域 (TAD),以改进基于CRISPR的基因激活系统 (CRISPRa). 这些新型TAD增强了合成生物学和治疗应用.
科学领域:
- 合成生物学 合成生物学
- 分子生物学分子生物学
- 基因调节 基因调节
背景情况:
- 可编程的DNA结合平台与工程事务激活域 (TAD) 结合,对于合成转录控制至关重要.
- 目前的基于CRISPR的转录激活 (CRISPRa) 技术通常使用大量或耐受性较差的TAD,限制了它们的应用.
研究的目的:
- 从人类机械敏感转录因子定义和优化最小的TAD.
- 开发强大而紧的交换激活模块和CRISPRa平台,用于增强合成转录控制.
主要方法:
- 从人类机械敏感转录因子中设计了最小的TAD.
- 构建了多方交易激活模块 (MSN,NMS,eN3x9) 和CRISPR-DREAM平台.
- 测试了CRISPR-DREAM的特异性,稳定性和跨细胞类型和调节位置的转录刺激.
- 评估了MSN和NMS在不同CRISPR系统,TAL效应器和指蛋白中的可移植性.
- 在重新编程人类纤维细胞和人类初级细胞类型中证明有效.
- 开发了双重和全合一的CRISPRa腺相关病毒 (AAV) 系统.
主要成果:
- 开发了强大而紧的交易激活模块 (MSN,NMS,eN3x9) 和CRISPR-DREAM平台.
- CRISPR-DREAM在哺乳动物细胞类型中显示出特异性和稳健性,有效地激活了各种调节位置.
- MSN和NMS模块在各种CRISPR系统,TAL效应器和指蛋白中展示了可移植性.
- 通过使用dCas9-NMS.成功地将人类纤维细胞重新编程成诱导的多能干细胞.
- 机械敏感转录因子TADs在人类初级细胞中被证明是有效且耐受良好.
- 设计的紧型CRISPR和AAV系统,用于双重和全合一的应用.
结论:
- 来自机械敏感转录因子的紧型人类TAD提供了强大且耐受良好的合成转录控制.
- 开发的交易激活模块和CRISPR-DREAM平台具有多功能性,可以在不同的系统中应用.
- 这些进步有助于改善生物医学应用的CRISPRa技术,包括细胞重编程和基因疗法.
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