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在Saccharomyces cerevisiae中的定量和模块化CRISPR/dCas9-dCpf1双重功能系统

Qing Feng¹, Xiaoyu Ning¹, Lei Qin²

¹Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, School of Chemistry and Chemical Engineering, Ministry of Industry and Information Technology, Beijing Institute of Technology, Beijing, China.

Frontiers in bioengineering and biotechnology

|November 29, 2023

概括

研究人员开发了一种使用Sp-dCas9和Fn-dCpf1的双重CRISPR激活/抑制 (CRISPRa/i) 系统,用于精确的酵母代谢工程. 这种双功能系统为生物技术应用提供了对基因表达的增强控制.

科学领域:

合成生物学合成生物学
分子生物学分子生物学
代谢工程是代谢工程.

背景情况:

克里斯普尔/dCas9和克里斯普尔/dCpf1系统为酵母代谢途径调节提供了潜力,但具有局限性.
需要一种补偿方法来克服个别系统的缺陷.

研究的目的:

构建和验证基于Sp-dCas9和Fn-dCpf1.1的双功能CRISPR激活/抑制 (CRISPRa/i) 系统.
为了实现对酵母基因表达的精确定量控制,用于代谢工程.

主要方法:

验证了酵母促进体的正交性和定量向,使用各种效应蛋白和RNA支架.
研究了CRISPR/dCas9和CRISPR/dCpf1系统的基因调节率和crRNA效率.
开发了一个正交的CRISPR/dCas9-dCpf1抑制系统,用于同时进行双基因调节.

主要成果:

克里斯普尔/dCas9系统实现了81.9%的抑制到627%的激活.
克里斯普尔/dCpf1系统表现出高达530%的转录抑制.
正角系统同时调节mCherry (54.6%) 和eGFP (62.4%) 没有交叉通话.

结论:

通过使用双功能CRISPR/dCas9-dCpf1系统建立了一家用于β-胡卜素生产的工程酵母细胞工厂.
该系统使得Saccharomyces cerevisiae中异质和内源代谢途径的向调节成为可能.
已证明在酵母生物技术中同时进行CRISPRa/i网络控制的卓越定量有效性和可扩展性.

关键词:

在CRISPR/dCas9-dCpf1中使用.这种植物是Saccharomyces cerevisiae.这是一个双功能系统系统.这是一个模块化的模块化系统.这是一个定量性的定量.乙-胡卜素 β-胡卜素的含量

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