Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9.
Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9.
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DOI：
10.1021/acssynbio.5b00147
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发表时间：
2016-01-15
影响因子：
4.7
通讯作者：
Tsimring L
中科院分区：
文献类型：
--
作者：
Didovyk A;Borek B;Hasty J;Tsimring L
合成基因回路开发的进展受到可用转录因子库有限的阻碍。最近，它已经使用CRISPR/Cas9系统大大扩展。然而，该系统受到其不完美的DNA序列特异性的限制，导致与宿主基因组或电路组件的潜在串扰。此外，CRISPR/Cas9介导的基因调控是环境依赖性的，影响基于Cas9的转录因子的模块化。在本文中，我们通过开发一种用于选择Cas9/gRNA转录因子/启动子对的计算方法来解决特异性和模块化的问题，所述Cas9/gRNA转录因子/启动子对彼此最大程度地正交，并且与宿主基因组和合成电路组件正交。我们通过设计和实验测试四个正交启动子/阻遏物对的背景下，一个强大的启动子PL从噬菌体λ的方法进行验证。我们证明，这些启动子可以通过构建一个双重和三重逆变器电路接口。为了解决模块化的问题，我们提出并实验验证了一种可预测地将正交CRISPR/Cas9调控纳入一大类天然启动子的方案。
The progress in development of synthetic gene circuits has been hindered by the limited repertoire of available transcription factors. Recently, it has been greatly expanded using the CRISPR/Cas9 system. However, this system is limited by its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent, affecting the modularity of Cas9 based transcription factors. In this paper we address the problems of specificity and modularity by developing a computational approach for selecting Cas9/gRNA transcription factor/promoter pairs that are maximally orthogonal to each other as well as to the host genome and synthetic circuit components. We validate the method by designing and experimentally testing four orthogonal promoter/repressor pairs in the context of a strong promoter PL from phage lambda. We demonstrate that these promoters can be interfaced by constructing a double and a triple inverter circuits. To address the problem of modularity we propose and experimentally validate a scheme to predictably incorporate orthogonal CRISPR/Cas9 regulation into a large class of natural promoters.