Reversal Of Mcr-1-mediated Colistin Resistance In Escherichia Coli By CRISPR-Cas9 System

Antibiotics have significant impacts on health care since their first introduction in the early 20 th century.However,with their irrational use in human medicine and animal husbandry,the emergence and prevalence of bacterial resistance has accelerated.Colistin is particularly important as the last-line refuge against multidrug-resistant Gram-negative bacteria infections.However,since the plasmid-mediated colistin resistance gene mcr-1 was first reported in 2015,the issue of colistin resistance has attracted widespread attention.Then mcr-1 gene has been detected all over the world.The prevalence of mcr-1 gene showed a global trend,which has brought great challenges to clinical treatment.Restoring colistin sensitivity is likely to improve therapeutic outcomes significantly,while the development of new antibiotics is very difficult and the combination of drugs has many drawbacks such as cross-resistance and bacterial imbalance.Therefore,exploring more efficient and specific methods to deal with the increasingly serious problem of bacterial resistance has become the focus of current researches.In recent years,CRISPR-Cas9,a widely used gene editing technology,is expected to be a new method to alleviate the problem of bacterial resistance.The purpose of this paper is to investigate whether CRISPR-Cas9 technology can cure the plasmid containing mcr-1 gene in Escherichia coli(E.coli),resensitize sensitivity to colistin and reduce the spread of mcr-1 gene.In this study,two lengths of sg RNAs(20 bp and 30 bp)specific to mcr-1 gene were designed and cloned into plasmid p Cas9,respectively.At the same time,we constructed colistin-resistant E.coli containing high-copy plasmid p UC19-mcr-1 or wild-type plasmid p HNSHP45.The engineered CRISPR-Cas9 system was transformed into E.coli carrying p UC19-mcr-1 or p HNSHP45,separately.The elimination efficiency was evaluated by PCR and quantitative real-time PCR(q PCR).The antimicrobial susceptibility test was performed using the broth microdilution method.The results showed that the engineered CRISPR-Cas9 system could efficiently eliminate mcr-1-harboring plasmids.For the wild-type plasmid p HNSHP45,the elimination rate was 75% in the selected 24 strains;for high-copy plasmid p UC19-mcr-1,the elimination rate was 100% in the selected 24 strains.The minimum inhibitory concentration of the knockout strain was reduced to 0.25 μg/m L compared to wildtype strain(2 μg/m L).For the high-copy plasmid p UC19-mcr-1,elimination efficiency can be achieved 50% in 6 hours and 80% in 8 hours.We found that there was no significant correlation between sg RNA lengths and elimination efficiency,while the content of plasmid backbone had an influence on efficiency.Furthermore,the conjugation assays verified that the engineered CRISPR-Cas9 system in bacteria can protect the recipient from plasmid-borne mcr-1 transfer via conjugation.Finally,sequence analysis showed that three different unexpected mutations occurred in CRISPR-Cas9 system,which caused the structure incomplete and made this system less effective on eliminating the mcr-1 gene.In conclusion,we presented a method that only one plasmid-mediated CRISPR-Cas9 system can be used to efficiently eliminate mcr-1 gene and resensitize E.coli to colistin.Moreover,this system provided a great potentiality to counteract the horizontal transmission of mcr-1 among bacterial pathogens.Hence,the CRISPR-Cas9 system could be used as a new method to control the spread of bacterial resistance,but it is still necessary to optimize its delivery method for clinical treatment in the future.