CRISPR Targeting And Incising Toxin Gene TcdB Inhibits The Growth Of Toxigenic Clostridioide Difficile

Objective: The CRISPR/Cas9 system was used to cleave the major virulence factor tcdB gene of Clostridioide difficile to achieve specific inhibition the growth of toxigenic Clostridioide difficile.Method: The sgRNA targeting tcdB was designed online,and sgRNA and Cas9 were separately or jointly acted on the tcdB gene amplification product to verify the cleavage efficiency in vitro.Penetrating peptide was used to connect sgRNA and Cas9 to improve the delivery efficiency.CPP-sgRNA,CPP-Cas9,CPP,CPP-sgRNA+CPP-Cas9,sgRNA+CPP-Cas9 were applied to Clostridium difficile producing toxin respectively,and the inhibition effect on bacterial growth was observed by monitoring the OD600 value of the bacterial liquid and the colony count.The same method was used to treat other pathogens 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 blank control,the groups in whichCPP-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;the group in which sgRNA and CPP-Cas9 acted together,it has a slightly inhibited effect on bacterial growth.After treated with CPP-sgRNA+CPP-Cas9 by the same method,there was no difference of the OD600 value and the colony count between the blank control and the non-toxic Clostridium difficile,Escherichia coli,Pseudomonas aeruginosa,Klebsiella pneumoniae,Staphylococcus aureus,Enterobacter cloacae and Acinetobacter baumannii.Conclusion: The sgRNA targeting tcdB established in this study combined with Cas9 can efficiently cleave the tcdB gene and inhibit the growth of pathogenic Clostridium difficile in vitro.CPP can improve the efficiency of sgRNA transfecting into bacteria.