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.
Keywords: CRISPR/dCas9; Escherichia coli; Gene knock-down; Gene overexpression; Toxic gene.
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