基于靶向多拷贝基因sgRNA表达盒替换的一种通用反选择技术

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

摘要:

筛选标记作为基因编辑中的重要工具，其应用范围常因宿主遗传背景及代谢兼容性差异而呈现种属特异性限制。为了解决这一问题，本研究利用CRISPR/Cas9系统开发了一种通用性更强的反选择工具，设计引入sgRNA表达盒作为反选择标记，引导Cas9蛋白靶向切割基因组DNA，对sgRNA表达盒替换的菌株进行筛选。该系统经过优化将sgRNA靶向多拷贝位点，增强了细胞致死率，从而将反选择效率提高到85.00%以上。这一反选择工具不受菌种的限制，应用范围更广，适用于多拷贝的质粒组装以及质粒编辑等各种场景。

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.