Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi).
Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi).
复制标题
DOI：
10.1007/978-1-4939-2687-9_23
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Qi LS
中科院分区：
文献类型：
--
作者：
Hawkins JS;Wong S;Peters JM;Almeida R;Qi LS
重复的规则散布的短回文重复序列（CRISPR）干扰（CRISPRi）是一种用于序列特异性抑制细菌细胞中基因表达的强大技术。CRISPRi只需要一种蛋白质和一种定制的指导RNA，就可以实现特定的基因靶向。在大肠杆菌中，CRISPRi阻遏效率很高（约300倍），并且没有观察到脱靶效应。该方法可以作为使用多种设计的指导RNA同时抑制许多基因的一般策略来扩大规模。在这里，我们提供了一个有效的指导RNA设计，克隆和CRISPRi系统在大肠杆菌中的测定方案。杆菌原则上，该方案可用于构建CRISPRi系统，用于其他细菌物种中的基因抑制。
Clustered regularly interspersed short palindromic repeats (CRISPR) interference (CRISPRi) is a powerful technology for sequence-specifically repressing gene expression in bacterial cells. CRISPRi requires only a single protein and a custom-designed guide RNA for specific gene targeting. In Escherichia coli, CRISPRi repression efficiency is high (~300-fold), and there are no observable off-target effects. The method can be scaled up as a general strategy for the repression of many genes simultaneously using multiple designed guide RNAs. Here we provide a protocol for efficient guide RNA design, cloning, and assay of the CRISPRi system in E. coli. In principle, this protocol can be used to construct CRISPRi systems for gene repression in other species of bacteria.