利用CRISPR/Cas9系统抑制Kan耐药基因水平转移研究

摘要

Objective To explore an effective technique to control the horizontal transfer of drug-resistance genes using specific CRISPR/Cas9 system.Methods Three pairs of kanamycin-resistance gene (Kan)-specific single guide RNAs (sgRNAs) were designed, annealed and inserted into the Bsa Ⅰ site of pCas9, respectively.Each group of recombinant bacteria competent cells were transformed by pET-30a that carried Kan gene.The transformants were spread on plates containing chloramphenicol or chloramphenicol/kanamycin to calculate and compare the transformation efficiencies.The DH5α competent cells containing pET-30a were transformed with each recombinant plasmid to detect the degradation of Kan gene mediated by specific sgRNAs with PCR and to observe the effects on growth curve of recombinant.Results The transformation efficiencies of Kan gene into Escherichia coli strains harboring specific sgRNAs CRISPR/Cas9 system decreased 100%-88.08%.The specific sgRNAs CRISPR/Cas9 system degraded Kan gene without affecting the growth of bacteria.Conclusion The specific CRISPR/Cas9 system inhibits the horizontal transfer of bacterial kanamycin-resistance gene.%目的 研究细菌耐药基因水平转移的控制技术.方法 以卡那霉素耐药基因Kan为靶基因设计3组靶向sgRNA,分别退火后插入到pCas9质粒中,重组菌用氯化钙法制成感受态,将含有Kan耐药基因的质粒pET-30a向其转化,转化菌液分别涂布含氯霉素和含氯霉素/卡那霉素平板,比较各组转化效率.将携带Kan特异性sgRNA的重组质粒分别转化含pET-30a的大肠埃希菌,PCR检测Kan耐药基因的降解,并绘制转化子在氯霉素平板上的生长曲线以分析特异性sgRNA对受体菌生长的影响.结果 所设计的3组sgRNA中有2组可以介导CRISPR/Cas9系统对Kan耐药基因水平转移的完全抑制,其余1组也可使转移效率降低88.08%.抑制机制为特异性CRISPR/Cas9系统降解了Kan耐药基因.未发现3组sgRNA对受体菌生长有明显影响.结论 特异性CRISPR/Cas9系统可抑制细菌Kan耐药基因的水平转移.