SGRN: A Cas12a-driven Synthetic Gene Regulatory Network System.
SGRN: A Cas12a-driven Synthetic Gene Regulatory Network System.
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SGRN：Cas12a 驱动的合成基因调控网络系统。
DOI：
10.1101/2023.05.08.539911
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Cusanovich,DarrenA
中科院分区：
文献类型：
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作者：
Kang,HyunJin;Fitch,JohnC;Varghese,ReebaP;Thorne,CurtisA;Cusanovich,DarrenA
基因调控网络控制基因在发育过程中的表达模式和对刺激的反应，使用调控逻辑模块来协调输入和输出。调控逻辑模块的一个例子是基因调控级联（GRC），其中一系列转录因子基因按顺序启动。合成生物学家已经推导出编码调控规则的人工系统，包括GRCs。此外，单细胞方法的发展使得在各种实验环境中发现基因调控模块成为可能。然而，可用于验证这些观察结果的工具仍然有限。在DNA切割缺陷Cas9 （dCas9）合成GRC的基础上，我们设计并实现了DNA切割缺陷Cas12a （dCas12a）合成GRC的替代方案。比较这两种系统表达荧光报告基因的能力，dCas9系统最初更活跃，而dCas12a系统更精简。研究了系统中单个组件的影响，确定了核定位是活性差异的主要驱动因素。与dCas9系统相比，dCas12a系统核定位的改善导致报告基因阳性细胞增加1.5倍，报告基因强度增加15倍。我们把这个优化的系统称为“合成基因调控网络”（SGRN，发音为“sojourn”）。
Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the “Synthetic Gene Regulatory Network” (SGRN, pronounced “sojourn”).