一个可编程的,无选择的CRISPR干扰系统在金黄色葡萄球菌中用于长期宿主相互作用研究
Roni Miah1, Mona Johannessen1, Morten Kjos2
1Department of Medical Biology and Centre for New Antibacterial Strategies (CANS), UiT- The Arctic University of Norway, 9019 Tromsø, Norway.
iScience
|September 22, 2025
概括
研究人员开发了一种针对 Staphylococcus aureus 的无选择CRISPR干扰 (CRISPRi) 系统. 这种新的系统可以在没有抗生素或诱导剂的情况下使基因沉默,从而推进细菌病原体研究.
科学领域:
- 微生物学和分子生物学
- 细菌遗传学和病变发生学
- 基因调控基于CRISPR的基因调控
背景情况:
- 基于dCas9的传统CRISPR干扰 (CRISPRi) 系统需要抗生素选择和外部诱导剂,限制它们在感染模型中的使用.
- 黄金菌 (Staphylococcus aureus) 是一个重要的人类病原体,需要精确的工具来研究其毒性因素和病原性.
- 开发无抗生素和无诱导剂的基因操纵工具对于推进复杂生物系统研究至关重要.
研究的目的:
- 为了设计一种新的,无选择的CRISPR干扰 (CRISPRi) 系统,用于*金黄色葡萄球菌*.
- 为了实现可编程的基因沉默在金黄色菌,而不需要抗生素耐药性标记物或外源诱导分子.
- 为了验证该系统在细菌病变发生的长期基因功能研究中的有效性.
主要方法:
- 开发一种CRISPRi系统,利用S. aureus*中稳定的pCM29等离子体.
- 在内源性促进体下整合dCas9表达,在构成性促进体下整合sgRNA表达,以实现无选择和无诱导体的操作.
- 编程系统以向凝结酶和自解酶基因,然后使用qPCR和表型分析 (体外和体外感染模型) 进行验证.
主要成果:
- 在黄金菌中成功建立了无选择的CRISPRi系统.
- 在没有抗生素压力的情况下,经过27代以上的稳定基因沉默.
- 通过qPCR确认了目标基因敲除,并观察到等离子体凝血和感染模型 (THP-1细胞, *Galleria mellonella *) 的相应的表型变化.
结论:
- 开发的CRISPRi系统提供了一个强大的,多功能工具,用于对金黄色葡萄球菌*进行基因操纵.
- 这种无选择和无诱导体的系统显著增强了对细菌基因功能的研究,特别是在长期的病原体研究中.
- 该系统作为创建其他细菌物种中类似的CRISPRi工具的基础蓝图.
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