Background

The cloning of toxic genes in E. coli requires strict regulation of the target genes' leaky expression. Many methods facilitating successful gene cloning of toxic genes are commonly exploited, but the applicability is severely limited.

Methods

A CRISPR/dCas9-assisted system was used to clone toxic genes in E. coli. The plasmid-based and genome-integrated systems were designed in this study. And the green fluorescent protein characterization system was used to test the repression efficiency of the two systems.

Results

We optimized the plasmid-based CRISPR/dCas9-assisted repression system via testing different sgRNAs targeting the Ptrc promoter and achieved inhibition efficiency up to 64.8%. The genome-integrated system represented 35.9% decreased GFP expression and was successfully employed to cloned four toxic genes from Corynebacterium glutamicum in E. coli.

Conclusions

Using this method, we successfully cloned four C. glutamicum-derived toxic genes that had been failed to clone in conventional ways. The CRISPR/dCas9-assisted gene cloning method was a promising tool to facilitate precise gene cloning of different origins in E. coli.

General significance

This system will be useful for cloning toxic genes from different origins in E. coli, and can accelerate the related research of gene characterization and heterologous expression in the metagenomic era.

中文翻译：

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在大肠杆菌中克隆有毒基因的 CRISPR/dCas9 辅助系统

背景

在大肠杆菌中克隆有毒基因需要严格调控靶基因的泄漏表达。许多有助于成功克隆有毒基因的方法通常被利用，但适用性受到严重限制。

方法

使用 CRISPR/dCas9 辅助系统在大肠杆菌中克隆有毒基因。本研究设计了基于质粒和基因组整合的系统。并使用绿色荧光蛋白表征系统来测试两种系统的抑制效率。

结果

我们通过测试针对 P trc启动子的不同 sgRNA 优化了基于质粒的 CRISPR/dCas9 辅助抑制系统，并实现了高达 64.8% 的抑制效率。基因组整合的系统中表示35.9％降低的GFP表达，并成功地用于从克隆的4个毒性基因的谷氨酸棒杆菌中的大肠杆菌。

结论

使用这种方法，我们成功克隆了四个传统方法未能克隆的谷氨酸棒杆菌衍生的毒性基因。CRISPR/dCas9 辅助的基因克隆方法是促进大肠杆菌中不同来源的精确基因克隆的有前途的工具。

一般意义

该系统将有助于在大肠杆菌中克隆不同来源的有毒基因，并可以加速宏基因组时代基因表征和异源表达的相关研究。