State Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, P.R. China.
Nucleic Acids Res. 2022 Jun 10;50(10):5988-6000. doi: 10.1093/nar/gkac463.
Saccharomyces cerevisiae is an important model eukaryotic microorganism and widely applied in fundamental research and the production of various chemicals. Its ability to efficiently and precisely control the expression of multiple genes is valuable for metabolic engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)-mediated regulation enables complex gene expression programming; however, the regulation efficiency is often limited by the efficiency of pertinent regulators. Here, we developed CRISPR-mediated protein-tagging signal amplification system for simultaneous multiplexed gene activation and repression in S. cerevisiae. By introducing protein scaffolds (SPY and SunTag systems) to recruit multiple copies of regulators to different nuclease-deficient CRISPR proteins and design optimization, our system amplified gene regulation efficiency significantly. The gene activation and repression efficiencies reached as high as 34.9-fold and 95%, respectively, being 3.8- and 8.6-fold higher than those observed on the direct fusion of regulators with nuclease-deficient CRISPR proteins, respectively. We then applied the orthogonal bifunctional CRISPR-mediated transcriptional regulation system to regulate the expression of genes associated with 3-hydroxypropanoic acid production to deduce that CRISPR-associated regulator recruiting systems represent a robust method for simultaneously regulating multiple genes and rewiring metabolic pathways.
酿酒酵母是一种重要的模式真核微生物，广泛应用于基础研究和各种化学品的生产。其高效、精确地控制多个基因表达的能力在代谢工程中具有重要价值。簇状规律间隔短回文重复序列（CRISPR）介导的调控可实现复杂的基因表达编程；然而，调控效率通常受到相关调控因子效率的限制。在这里，我们开发了 CRISPR 介导的蛋白标记信号放大系统，用于在酿酒酵母中同时进行多重基因激活和抑制。通过引入蛋白支架（SPY 和 SunTag 系统）将多个调控因子招募到不同的无核酸酶 CRISPR 蛋白，并进行设计优化，我们的系统显著提高了基因调控效率。基因激活和抑制效率分别高达 34.9 倍和 95%，分别比直接将调控因子与无核酸酶 CRISPR 蛋白融合观察到的效率高 3.8 倍和 8.6 倍。然后，我们将正交双功能 CRISPR 介导的转录调控系统应用于调节与 3-羟基丙酸生产相关的基因的表达，推断出 CRISPR 相关调控因子招募系统代表了一种同时调控多个基因和重新布线代谢途径的强大方法。
