为可扩展和多输入的CRISPRa/i电路设计可激活的促进器.
Diego Alba Burbano1,2, Ryan A L Cardiff2,3, Benjamin I Tickman2,3
1Department of Chemical Engineering, University of Washington, Seattle, WA 98195.
概括
科学家们使用CRISPR技术设计了高性能可激活促进体,用于构建复杂的基因调节网络 (GRNs). 这一突破使得在无细胞系统中具有精确控制的更深更宽的合成GRNs成为可能.
科学领域:
- 合成生物学 合成生物学
- 分子生物学分子生物学
- 生物技术是生物技术.
背景情况:
- 基因调节网络 (GRNs) 是生物系统的基础.
- 基于CRISPR的基因电路为构建合成GRNs提供了一个强大的平台.
- 开发高性能组件对于先进的GRN工程至关重要.
研究的目的:
- 为CRISPR激活和干扰 (CRISPRa/i) 基因调控网络开发一种创造高性能可激活促进体的方法.
- 为了实现深度,宽度和多输入合成GRNs的构建.
- 证明工程促进剂在无细胞系统中的通用性和应用.
主要方法:
- 基于序列的综合设计和体内查,以设计可激活的促进体.
- 利用以大肠杆菌为基础的无细胞系统进行促进体的表征.
- 在多层CRISPRa/iGRN中组装工程发起物.
主要成果:
- 在工程可激活促进器中达到高达1000倍的动态范围.
- 演示了CRISPRa GRNs的构建,六层深,四个分支宽.
- 改善了EL222光遗传系统的动态范围,从6倍提高到34倍.
- 在小分子和蛋白质-蛋白质相互作用介导的CRISPRa系统中展示了应用.
结论:
- 开发了一种可通用的方法来设计高动态范围可激活的促进剂.
- 在无细胞系统中启用了新的基因调控功能,包括反循环和逻辑门.
- 提升了基于CRISPR的合成生物学潜力,用于复杂的生物电路构造.
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