Construction Of The CRISPR-Cas9 Vector System Targeting OXA23 Gene

Objective:In this study,the plasmid pet41a was used as the vector backbone,and the target gene fragment was obtained from the plasmid pwt-Cas9 and pgRNA by double enzyme digestion,and the CRISPR-Cas9 vector system was constructed to establish an effective gene editing vector for studying bacterial drugs resistance.using thermal conversion and electroporation two molec?lar techniques to prepare competcnt cells and optimize transformation conditions to obtain maximum conversion efficiency.Methods:1.Collect 105 strains of Acinetobacter baumannii isolated from the General Hospital of Ningxia Medical University from 2016 to 2017.2.Drug sensitivity test and PCR technique were used to screen Acinetobacter baumannii strains that sensitive to kanamycin and containing OXA23 gene.3.The plasmids pet41a,pgRNA and pwt-Cas9were selected and analyzed the acid sequence and restriction sites.The plasmids pet41a and pwt-Cas9 were digested with restriction endonucleases XhoI and BgiII,to obtain linear plasmid fragments with the same cohesive ends,which were ligated with T4 DNA ligase,and ligated products were transformed into DH5?competcnt cells.The solid LB medium of kanamycin was subjected to positive monoclonal screening and squencing,and the constructed plasmid was named pet41-Cas9.Pet41-Cas9 and pgRNA were digested with BgiII and XbaI restriction enzymes.After purification,ligation and transformation,positive monoclonal colonies were screened and sequenced.The recombinant plasmid was named pet41a-Cas9-sgRNA.The recombinant plasmids pet41-Cas9 and pet41a-Cas9-sgRNA were transformed into BL-21(DE3),and the proteins were extracted by IPTG for SDS-PAGE.4.Acinetobacter baumannii competcnt cells were prepared with deionized water and calcium chloride respectively,and transfection by electricity and heat into standard strain ATCC17978Res?lts:Two strains sensitive to kanamycin and containing the OXA23 gene were screened from clinical and named as AB7 and AB34.Sequencing confirmed that the plasmids pet41-Cas9 and pet41a-Cas9-sgRNA were correctly linked and the vector was successf?lly constructed.The recombinant plasmid was transfected into DE3 and induced by IPTG for 4 h,and the recombinant protein with molec?lar mass unit of 160×10~3 was expressed,which was consistent with the theoretical molec?lar mass of Cas9 protein.Competcnt cells prepared by calcium chloride method have positive transforming strains under thermotransfection conditions,and the competcnt cells prepared by deionized water has only positive transforming strains under electrotransfection.At different voltage transfections,positive transforming bacteria appeared at 1000v,and with the higher of the voltage,the more colonies were grown.Conclusion:1.Successf?l construction of CRISPR-Cas9 vector system plasmid pet41a-Cas9-sgRNA and expression of recombinant Cas9 protein,laid the experimental basis for the editing of bacterial genes.2.The transfection efficiency of competcnt cells prepared by calcium chloride method was slightly higher than competcnt cells by deionized water method;the electrotransfection efficiency was significantly higher than that of thermal transfection,and the transfection efficiency increased with the increase of voltage.The maximum is reached at 2000v.