CRISPR靶向切割毒力因子tcdB基因抑制致病艰难梭菌生长

CRISPR targeting and incising toxin gene tcdB inhibits the growth of disease-causing Clostridioide difficile

目的应用CRISPR/Cas9系统对艰难梭菌主要毒力因子tcdB基因进行切割,达到特异性抑制致病艰难梭菌生长的效果。方法在线设计针对tcdB的sgRNA,分别将sgRNA与Cas9单独或共同作用于tcdB基因扩增产物,验证体外切割效能;分别将穿膜肽(CPP)-sgRNA或单独的sgRNA与CPP-Cas9单独或共同作用于产毒艰难梭菌,通过监测菌液A值和菌落计数,观察对细菌生长的抑制效果。同样的方法处理肠道其他细菌,验证特异性。结果sgRNA和Cas9共同作用可把tcdB扩增产物有效切割,而单独加入sgRNA或Cas9均无切割作用;单独加入CPP-sgRNA、CPP-Cas9、CPP对细菌生长没有影响;CPP-sgRNA与CPP-Cas9共同作用,无细菌生长;单独sgRNA与CPP-Cas9共同作用,细菌生长轻度受抑。用同样的方法将CPP-sgRNA+CPP-Cas9处理肠道其他细菌后,菌液A值和菌落计数与空白对照无差别。结论建立的针对tcdB的sgRNA与Cas9混合可在体外高效切割tcdB基因,抑制细菌生长;CPP可提高sgRNA转染进入细菌的效率。

Objective The CRISPR/Cas9 system was used to cleave the major virulence factor tcdB gene of Clostridioide difficile to achieve specific inhibition of the growth of disease-causing Clostridioide difficile.Methods The sgRNA targeting tcdB was designed online,and sgRNA and Cas9 were separately or co-operated on the tcdB gene amplification product to verify the cleavage efficiency in vitro.The CPP-sgRNA or the sgRNA alone and CPP-Cas9 were used alone or in combination to the toxin-producing Clostridioide difficile,the inhibition of bacterial growth was observed by monitoring the A value of the bacterial liquid and the colony count.The same method was used to treat other bacteria in the intestine for specific verification.Results The combination of sgRNA and Cas9 can effectively cleave the tcdB amplification product,while sgRNA or Cas9 alone has no cleavage effect.In contrast to the untreated group,the groups in which CPP-sgRNA,CPP-Cas9,and CPP were added alone had no effect on bacterial growth;the group in which the CPP-sgRNA interacted with CPP-Cas9 has no bacterial growth;sgRNA and CPP-Cas9 Together,it has a slightly inhibited effect on bacterial growth.After treated used CPP-sgRNA+CPP-Cas9 by the same method,there was no difference of the A value and the colony count of the other intestinal bacteria from the blank.Conclusion The sgRNA targeting tcdB combined with Cas9 can efficiently cleave the tcdB gene and inhibit bacterial growthin in vitro.CPP can improve the efficiency of sgRNA transfecting into bacteria.