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化学诱导的CRISPR/Cas9电路用于超高动态范围的基因扰动

Rajini Srinivasan¹, Tao Sun¹, Azalia Sandles¹

¹Department of Molecular Biology, Genentech Inc., South San Francisco, CA, USA.

Nature communications

|December 12, 2025

概括

研究人员开发了改进的CRISPR/Cas9系统,用于细胞中精确的基因编辑. 这些新工具可以更好地控制Cas9的活动,尽量减少意外的编辑,并提高疾病建模的实验可靠性.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
细胞工程细胞工程

背景情况:

对于疾病建模和基因功能研究来说,CRISPR/Cas9是至关重要的.
现有的Tet-On系统表现出泄漏的Cas9表达和微弱的诱导活性.
泄漏的Cas9可以导致累积的DNA损伤和基因组退化.

研究的目的:

开发具有改进控制的先进的可诱导CRISPR/Cas9系统.
克服现有系统的局限性,例如漏水和弱感应.
为了提高Cas9介导基因编辑的动态范围和可靠性.

主要方法:

工程转基因平台结合了有条件的Cas9破坏稳定和抑制.
开发了一种全合一的,超紧密的Tet可诱导的CRISPR系统.
创建了一个 Branaplam 调节的拼接开关模块,用于替代感应.
构建了一个双控制,Tet诱导的CRISPR干扰 (CRISPRi) 模块.

主要成果:

在OFF状态下实现了最小化的Cas9功能,并最大化了ON状态的效率.
在各种细胞系和目标中显示出异常的动态范围 (ON vs. OFF状态).
使用Branaplam系统验证了强大的Cas9活动控制与低基线.
通过CRISPRi模块展示了强大的转录沉默.

结论:

新一套可诱导的CRISPR系统提供了卓越的控制和效率.
这些先进的工具在各种细胞类型和实验环境中具有广泛的适用性.
开发的系统使得更精确的疾病建模和基因功能分析成为可能.

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