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基于CRISPRi的贝氏柯克斯体dotB基因沉默

SILENCING OF COXIELLA BURNETII DOTB BASED ON CRISPR INTERFERENCE
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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。 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.
作者 周春雨 赵明亮 程如熹 张珊 李娜娜 于永慧 欧阳譞 焦俊 熊小路 张家宁 ZHOU Chun-yu;ZHAO Ming-liang;CHENG Ru-xi;ZHANG Shan;LI Na-na;YU Yong-hui;OUYANG Xuan;JIAO Jun;XIONG Xiao-lu;ZHANG Jia-ning(School of Public Health,Mudanjiang Medical University,Mudanjiang 157000,Heilongjiang,China;State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,AMMS,AMS,Beijing 100071,China)
出处 《寄生虫与医学昆虫学报》 CAS 2024年第1期6-11,共6页 Acta Parasitologica et Medica Entomologica Sinica
基金 国家自然科学基金面上项目(32370203)。
关键词 贝氏柯克斯体 沉默 CRISPRi dCas9 DotB Coxiella burnetii Silencing CRISPRi dCas9 DotB
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