Department of Biomedical Engineering, University of California Davis, Davis, CA 95616, USA.
Nucleic Acids Res. 2020 Jul 27;48(13):e76. doi: 10.1093/nar/gkaa451.
The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.
基因表达噪声的控制对于改善药物治疗和合成生物系统的性能非常重要。以前的工作通过改变转录起始、mRNA 降解和 mRNA 翻译的速度来调节基因表达噪声。然而，这些方法具有侵入性：它们需要改变目标遗传成分。在这里，我们基于 CRISPR-dCas9 创建了一个正交系统来调节基因表达噪声。具体来说，我们通过在单个细胞中同时整合 CRISPR 激活和抑制（CRISPRar）来调节大肠杆菌中报告基因的基因表达噪声。CRISPRar 使用单个识别两个不同单链向导 RNA（sgRNA）的 dCas9。我们构建了一个具有不同表达激活和抑制强度的 sgRNA 变体文库。我们发现 CRISPRar 可以独立地调节报告基因的表达噪声和平均值。我们的结果表明，表达噪声是通过两种 sgRNA 之间的竞争来调节的，这两种 sgRNA 调节 RNA 聚合酶与启动子的结合。CRISPRar 可能会改变我们在基因组水平上调节表达噪声的方式。我们的工作对基因功能、表型异质性和遗传电路控制的研究具有广泛的影响。
