合成基因电路结合了CRISPR干扰和CRISPR激活在E. coli:平等指导RNA结合 afinities 的重要性,以避免上下文依赖的效应
Içvara Barbier1, Hadiastri Kusumawardhani1, Lakshya Chauhan1,2
1Department of Fundamental Microbiology, University of Lausanne, 1015 Lausanne, Switzerland.
ACS synthetic biology
|October 9, 2023
概括
在合成生物学中,将CRISPR干扰 (CRISPRi) 和CRISPR激活 (CRISPRa) 结合起来可以产生不可预测的结果. 对于CRISPRi和CRISPRa复合体,使用统一的支架指导RNA结构可以防止在大肠杆菌中出现这些依赖上下文的效应.
科学领域:
- 合成生物学 合成生物学
- 基因表达调节 基因表达调节
- 在CRISPR技术的应用中.
背景情况:
- 对于 prokaryotic 的合成基因电路,已经确立了 CRISPR 干扰 (CRISPRi).
- 克里斯普尔激活 (克里斯普尔a) 尚未成熟,并且结合的克里斯普尔i/克里斯普尔a电路正在出现.
- Prokaryotic 基因电路设计需要对基因表达进行精确的控制.
研究的目的:
- 研究结合CRISPRi和基于SoxS的CRISPRa在大肠杆菌中的作用.
- 确定在双CRISPR系统中实现可预测行为的策略.
- 使用CRISPR工具优化合成基因电路的构建.
主要方法:
- 在大肠杆菌中共同表达CRISPRi和基于SoxS的CRISPRa系统.
- 使用不同的支架指导RNA结构用于CRISPRa和CRISPRi复合体.
- 对基因表达结果的比较分析与统一与不同的支架指导RNAs.
主要成果:
- 将CRISPRi与基于SoxS的CRISPRa结合起来,导致了取决于上下文的效应和可预测行为的丧失.
- 克里斯普拉和克里斯普拉i复合体形成亲缘关系的差异导致了不可预测的结果.
- 在CRISPRa和CRISPRi复合体中使用相同的支架导向RNA结构可以减轻这些影响.
结论:
- 选择支架指导RNA结构对于在联合CRISPRi/CRISPRa合成基因电路中的可预测性能至关重要.
- 一个统一的支架指导RNA方法可以克服与不同复杂亲和关系相关的局限性.
- 这一发现推动了使用CRISPR技术的强大和可预测的合成基因电路的设计原则.
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