Targeted Elimination of blaNDM-5 Gene in Escherichia coli by Conjugative CRISPR-Cas9 System

Purpose: Plasmid-borne carbapenem resistance gene blaNDM-5 accelerates the dissemination of carbapenem-resistant Enterobacteriaceae. To efficiently eliminate the blaNDM-5-harboring plasmid and sensitize the antibiotic-resistant bacteria to meropenem, we used the CRISPR-Cas9 system for combating the carbapenem-resistant Escherichia coli (E. coil). Methods: A series of CRISPR-Cas9 plasmids was constructed, and specific guide RNAs(sgRNA) were designed to target the blaNDM-5 gene. We used chemically transformation or conjugation delivery methods, and the elimination efficiency in each recipient strains was evaluated by plate counting, PCR and quantitative real-time PCR (qPCR). Antimicrobial susceptibility test was carried out by using the broth microdilution method. In addition, we assessed the effect of the CRISPR-Cas9 system of adaptive immunity on the prevention of the exogenous resistant plasmids pNDM-5 by introducing the system into E coli J53. Results: The results showed that pCas9, pCas9-oriT and pBAD-Cas9-oriT can effectively eliminate blaNDM-5 in E. coli with >94.00% elimination efficiency. The blaNDM-5-harboring E. coli successfully restored their susceptibility to meropenem, with eight-fold reduction of minimum inhibitory concentration (MIC) values (from 16 mu g/mL to 0.06 mu g/mL). The E. coli J53 strain containing Conclusion: The CRISPR-Cas9 system achieved plasmid clearance and simultaneous re-sensitization to meropenem in E. coli. The CRISPR-Cas9 system could block the horizontal transfer of plasmid pNDM-5. The conjugative delivery of CRISPR-Cas9 provides a new tool for the removal of resistance plasmids and sensitize the recipient to carbapenem. It provides a therapeutic approach to counteract the propagation of blaNDM-5 gene among clinical pathogens.