CRISPR/dCas9-RpoD-Mediated Simultaneous Transcriptional Activation and Repression in Shewanella oneidensis MR-1
CRISPR/dCas9-RpoD-Mediated Simultaneous Transcriptional Activation and Repression in Shewanella oneidensis MR-1
复制标题
DOI：
10.1021/acssynbio.2c00149
复制
发表时间：
2022-05-24
影响因子：
4.7
通讯作者：
Cao, Yingxiu
中科院分区：
文献类型：
--
作者：
Chen, Yaru;Niu, Xiaolong;Cao, Yingxiu
电活性微生物的胞外电子传递是生物电化学系统广泛应用的主要因素。希瓦氏菌MR-1是研究EET的模型细胞之一，它与多种细胞活动有关。然而，由于缺乏转录激活工具，调控多个基因是劳动密集型和耗时的，这阻碍了提高EET效率在S. oneidensis。在这项研究中，我们开发了一种易于操作和多功能的调控工具，即同时成簇规则间隔短回文重复序列（CRISPR）介导的转录激活（CRISPRa）和干扰（CRISPRi）系统，用于在S. oneidensis。首先，筛选了大量的活化剂，并确定RpoD（sigma（70））为最佳活化剂。第二，有效激活范围被鉴定为转录起始位点上游190-216个碱基。第三，上调和下调是通过两个正交的单向导RNA靶向不同的位置来实现的。细胞分裂基因（minCDE）的激活和细胞毒性基因（SO_3166）的抑制同时实施，增加了2.5倍的功率密度，提高了2.9倍的偶氮染料的降解率。同时CRISPRa和CRISPRi系统能够同时进行多重遗传调控，为进一步推进EET机制的研究和在S. oneidensis。
Extracellular electron transfer (EET) of electroactive microorganisms (EAMs) is the dominating factor for versatile applications of bio-electrochemical systems. Shewanella oneidensis MR-1 is one of the model EAMs for the study of EET, which is associated with a variety of cellular activities. However, due to the lack of a transcriptional activation tool, regulation of multiple genes is labor-intensive and time-consuming, which hampers the advancement of improving the EET efficiency in S. oneidensis. In this study, we developed an easily operated and multifunctional regulatory tool, that is, a simultaneous clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation (CRISPRa) and interference (CRISPRi) system, for application in S. oneidensis. First, a large number of activators were screened, and RpoD (sigma(70)) was determined as the optimal activator. Second, the effective activation range was identified to be 190-216 base upstream of the transcriptional start site. Third, up- and downregulation was achieved in concert by two orthogonal single guide RNAs targeting different positions. The activation of the cell division gene (minCDE) and repression of the cytotoxic gene (SO_3166) were concurrently implemented, increasing the power density by 2.5-fold and enhancing the degradation rate of azo dyes by 2.9-fold. The simultaneous CRISPRa and CRISPRi system enables simultaneous multiplex genetic regulation, offering the potential to further advance studies of the EET mechanism and application in S. oneidensis.