基于CRISPRi的贝氏柯克斯体dotB基因沉默
SILENCING OF COXIELLA BURNETII DOTB BASED ON CRISPR INTERFERENCE
周春雨 1赵明亮 2程如熹 2张珊 2李娜娜 2于永慧 2欧阳譞 2焦俊 2熊小路 1张家宁2
作者信息
- 1. 牡丹江医学院公共卫生学院, 黑龙江牡丹江 157000;军事科学院军事医学研究院微生物流行病研究所, 病原微生物生物安全全国重点实验室, 北京 100071
- 2. 军事科学院军事医学研究院微生物流行病研究所, 病原微生物生物安全全国重点实验室, 北京 100071
- 折叠
摘要
目的 更简便快捷地研究贝氏柯克斯体基因功能.方法 基于CRISPRi原理构建携带化脓链球菌dCas9 的重组质粒,将靶向贝氏柯克斯体dotB基因的sgRNA序列插入重组质粒中并将质粒经电转化导入贝氏柯克斯体,利用脱水四环素(aTC)诱导dCas9 表达以抑制贝氏柯克斯体dotB基因的转录,从而建立基于CRISPRi系统的贝氏柯克斯体基因沉默技术.结果 在aTC诱导下,该CRISPRi系统中dCas9 可正常表达,dotB在转录水平和蛋白水平均被抑制,dotB表达抑制后贝氏柯克斯体胞内生长繁殖水平降低.结论成功建立起基于CRISPRi的贝氏柯克斯体的基因沉默技术,为研究贝氏柯克斯体特定基因的生物学功能研究提供了技术支撑.
Abstract
Objective A recombinant plasmid for CRISPR interference(CRISPRi)that incorporates dCas9 from Streptococcus pyogenes was constructed to investigate the function of Coxiella burnetii genes through a prompt,simple method.Methods A single guide RNA(sgRNA)sequence targeting C.burnetii dotB was integrated into a recombinant plasmid,which was then transformed into C.burnetii using electroporation.Next,dCas9 expression was induced with dehydrated tetracycline(aTC)to inhibit the transcription of dotB.Results The results demonstrated that dCas9 can be expressed normally in the CRISPRi system through aTC induction.Suppression of dotB was observed at the transcriptional and translational levels.In addition,the intracellular reproduction of C.burnetii significantly decreased after the suppression of dotB expression.Conclusion Silencing technology for the C.burnetii gene based on CRISPRi was established in this study,providing support for studying the biological functions of C.burnetii genes.
关键词
贝氏柯克斯体/沉默/CRISPRi/dCas9/DotBKey words
Coxiella burnetii/Silencing/CRISPRi/dCas9/DotB引用本文复制引用
基金项目
国家自然科学基金面上项目(32370203)
出版年
2024
国家科技期刊平台
NSTL
万方数据