CRISPR-Cas9 System-Based Gene Editing In The Escherichia Coli Genome And Bacterial Drug-Resistant Reversal

The discovery of antibiotic is the great milestone for fighting against the bacterial infection.However,the traditional antibiotic works by blocking conserved bacterial signaling or metabolic pathways,therefore it is unable to distinguish between the pathogen needed to be killed and the bacteria being good for us,Long-termed usage will lead to dysbacteriosis and drug-resistance.We need to find new antibacterial drugs that can target pathogens or drug-resistant bacteria specifically.CRISPR-Cas9 system is originated from bacterial adaptive immunity,which utilize the RNA and Cas-related protein to recognize the special DNA sequence and cut it.Compared with the traditional gene editing tools such as ZFNs and TALENs,it is more simple and effective.Thus CRISPR-Cas9 system,as a new gene editing tool,is applied in many fields for gene knock out,knock in or point mutant.On the other hand,researchers make use of it ability to recognizing gene sequence specially to distinguish pathogen or drug-resistant bacteria from normal bacterial flora in genetic level and to selectively eliminate pathogen and drug-resisant mutant.We utilized pCas and pTargetF,obtained from Shanghai Research Center of Industrial Biotechnology to expressed CRISPR-Cas9 system and successfully performed single gene(finA)deletion,simultaneous two-gene(ftnA and ftnB)deletion,and single base point utantion(in the kdpD gene)in E.coli genome.ftnA and ftnB encode iron storage related protein in E.coli genome.The efficiency for gene editing is almost 100%in the strains we test.For the purpose of eliminating drug-resistant bacteria,we constructed drug-resistant plasmids containing genes encoding carbapenemase,which confers resistance to?-lactams,and transformed it into WT(BW25113)to form drug-resistant bacteria.And then we designed N20-sgRNA targeting drug-resistant genes to clear drug-resistant bacteria specifically.Then,we reconstructed the two plasmid system into one plasmid system that only contains a new pCas-m plasmid to experss sgRNA under IPTP induced promoter.We used the new IPTG-induced system to observe the kinetic changes of clearing efficiency of drug-resistant genes.In order to improve the resistant gene elimination efficiency,we also made different tries,such as designing two different recognition and cut sites in the plasmid or using recombination repair-deficient strain.In order to expand the drug-resistant gene range that the CRISPR system could target,we tried to cleave clinically-isolated colistin-resistant plasmid.The result showed that we can eliminate drug-resistant plasmid containing target region specifically and resensitize bacteria to antibiotic.The maximal resistant gene removel efficiency can be as high as 6 orders of magnitude.Finally,in order to shorten the step and time when using CRISPR system,we tried to rebuild cloning-free CRISPR by using T7-promoter transcriptional system to simplify the plasmid construct process.We showed that the T7-promoter transcriptional system allow CRISPR system to function,but the result is not ideal because the segment sgRNA is not stable in the cell.So we need more ways to improve the stability of DNA fragment to achieve cloning-free's aim.