A universal counter-selection strategy based on replacement of sgRNA expression cassettes targeting multi-copy genes.

Qianru CAI; Manman WANG; Jinmei ZHU; Jiequn WU

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A universal counter-selection strategy based on replacement of sgRNA expression cassettes targeting multi-copy genes.

Author: Qianru CAI ¹ ; Manman WANG ¹ ; Jinmei ZHU ¹ ; Jiequn WU ¹
Author Information

1. Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China.
Publication Type:Journal Article
Keywords: Cas9-targeted cleavage; plasmid editing and assembly; scar-free editing; sgRNA counter-selection
MeSH: CRISPR-Cas Systems/genetics*; Gene Editing/methods*; RNA, Guide, CRISPR-Cas Systems/genetics*; Plasmids/genetics*
From: Chinese Journal of Biotechnology 2025;41(4):1649-1657
CountryChina
Language:Chinese
Abstract: Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.