A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast
A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast
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DOI：
10.1093/nar/gky1191
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发表时间：
2019-01-10
影响因子：
14.9
通讯作者：
Kolmar, Harald
中科院分区：
文献类型：
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作者：
Hofmann, Anja;Falk, Johannes;Kolmar, Harald
一个或多个感兴趣的基因的强健而精确的开关，然后是表达或抑制，对于许多生物电路以及工业应用都是必不可少的。然而，到目前为止已发表的许多调控系统影响宿主细胞的活性，表现出高基础表达或仅允许靶基因的过度表达，而不可能进行精细调控。在这里，我们描述了一种AND门，它结合了三个成熟系统的优点，即由Gal10作为天然转录因子控制的支架RNA CRISPR/dCas9平台和由LexA-ER-AD作为异源转录因子控制的与门。因此，我们开发了一个可预测的、模块化的、通用的表达控制系统。通过核糖体跳过T2a序列选择将感兴趣基因(GOI)与荧光团相结合的报告基因，允许系统在不丧失报告功能的情况下适应任何感兴趣的基因。为了更好地了解我们的系统的基本原理和功能，我们开发了一个数学模型和单细胞分析来支持我们的实验结果。
The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis.