Study On The Method Of Eliminating Multi-drug Resistance Gene Cfr In Escherichia Coli By Using CRISPR/Cas9 System

The development of antibiotic resistance in bacteria is one of the most serious threats to human health.The spread of antibiotic resistance genes is mainly resulted from the horizontal transfer including transformation,transduction and conjugation.Bacterial conjugation transfer is the most common and effective transmission mode which is mediated by self-transferable plasmids.Different sources of bacteria can be mediated by fthe plasmid to get the same antibiotic resistance spectrum,resulting in the clinical efficacy of antibiotics to reduce or even invalid.Plasmid mediated resistance is different from that caused by chromosomal mutations.Resistance plasmids can be spread by horizontal gene transfer,but once the resistance plasmid was eliminated,the resistance can not be restored unless exogenous resistance plasmids were transformed again.Therefore,it is of great significance to use molecular biology technology to eliminate drug resistance plasmids or to block the transmission of them.Cfr(chloramphenicol-florfeniclol resistance)gene is a multi-drug resistant gene,which is mostly carried by plasmids,and a few are located in chromosomes.The Cfr protein encoded by this gene is ribosomal 23 S rRNA methylase,which could simultaneously mediate drug resistance of five types of antimicrobial agents with different chemical structures,such as chloramphenicol,oxazolidinone,lincosamides,streptogramin a class and pleuromutilins.Currently,the cfr gene has been detected in many kinds of bacterial from animal sources and human sources in many areas,and it has a tendency to spread gradually.In this study,we made use of CRISPR/Cas9 technique to establish a highly efficient method,which can target specific gene cfr in Escherichia coli.First of all,we verified that the GFP reporter plasmid which containing Kanamycin resistance could be cleared by the CRISPR/Cas9 system in E.coli.It proved that the system can effectively eliminate the multiple copies of the plasmid by detecting the fluorescence and antibiotic resistance of GFP in E.coli.Secondly,using E.coli S17-1 as the donor strain,E,coli which carried cfr gene as the recipient bacteria,then we made a horizontal transmission of CRISPR/Cas9 system into the recipient bacteria clearing resistance plasmid through conjugation reaction,and the plasmid elimination rate was 0.22%.Finally,we constructed a self-transfer targeting vector,which could target multi-drug resistance gene cfr by conjugation reaction and it does not depend on the host strain S17-1.The plasmid clearance rate was 1,43%.In summary,the method which we had established not only can effectively eliminate resistance plasmids in recipient bacteria and restore the sensitivity to antibiotics,but also can immune to the target resistance gene.Thereby,this method can be used as a new tool for gene regulation across bacterial cells and potentially for bacterial population control.