Engineering Translational Activators with CRISPR-Cas System

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• 作者：Pei Du ; Chensi Miao ; Qiuli Lou ; Zefeng Wang ; Chunbo Lou
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：January 15, 2016
• 年：2016
• 卷：5
• 期：1
• 页码：74-80
• 全文大小：446K
• ISSN：2161-5063

文摘

RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility.