Study On PAgo Protein-mediated Bacterial Genome Editing And Its Mechanism

Although classic genetic manipulation tools had good effects on a few model bacterium,they were not applicable to other bacterium including most important pathogens.CRISPR/Cas9 was a new gene editing technology,which due to the presence of complex expression elements,high off-target efficiency,and the presence of anti-Cas9 molecules in bacterium and other factors,so those limited the application of this technology in many pathogens.Therefore,the development of universal,simple,efficient and seamless genetic manipulation tools is of great value and significance for the study on pathogenic mechanisms and prevention and control techniques of pathogens.Pasteurella multocida was seriously harmful to pig,cattle,chicken,duck and other livestock and poultry breeding industries.The lack of efficient genetic manipulation tools had greatly limited the study on pathogenic mechanism of the pathogen and the development of prevention and control products.In order to study on the pathogenic mechanism and genetic engineering vaccine of P.multocida,our laboratory applied a variety of classical genetic operating systems previously,such as thermo-sensitive system,Sac B selection system,gal K selection system and thymidylate synthase A selection system,etc.,to construct the mutant strains of avian pasteurella multocida,but no success.Then the target genes of avian Pasteurella multocida and rabbit Pasteurella multocida were successfully knocked out and knocked in by the NgAgo/gDNA system,and vaccine candidate strains with reduced virulence were obtained.The results indicated that the NgAgo/gDNA system provided a genetic manipulation tool for the study of the pathogenic mechanism and prevention and control products of P.multocida.But the molecular mechanism by which this system mediated bacterial genome editing was not clear.In view of this,this thesis mainly studied on the prokaryotic Argonaute(pAgo)protein mediated gene knockout of P.multocida and E.coli and its molecular mechanism,the research contents were as follows:1)NgAgo mediated the knock-out and knock-in of target genes in different bacterium.Previous studies in our laboratory had found that the NgAgo/gDNA system could effectively mediate the knockout and knock in of multiple genes in P.multocida,but whether this system could be used for gene knockout in other bacterium need further research.This study found that the efficiency of NgAgo/gDNA system in successfully mediating aste gene deletion on E.coli was as high as 90%.The results showed that the NgAgo/gDNA system could be applied for the gene knockout of bacterium such as P.multocida and E.coli.2)NgAgo mediated bacterial gene konckout independent of its own DNase activity.The CRISPR/Cas9 system enhanced homologous recombination effect by targeting genomic DNA with g RNA to achieve bacterial genome editing.Whether the NgAgo/ gDNA system-mediated bacterial gene knockout also depend on the hydrolase activity of gDNA targeted cleavage of genomic DNA needed further study.Therefore,we mutated the potential enzyme active sites(D633A,D738A)of NgAgo,and found that it could still efficiently mediate the knockout of lyi gene on avian Pasteurella multocida.NgAgo could still efficiently mediate the knockout of lyi and opa gene on avian Pasteurella multocida without the introducing exogenous gDNA.It shows that NgAgo improved the efficiency of bacterial gene knockout independent of its own DNase activity and exogenous gDNA.3)NgAgo protein improved homologous recombination efficiency by enhancing recA-mediated homologous DNA strand exchange.Since NgAgo-mediated bacterial gene knockout was independent of its own DNase activity and exogenous gDNA,the mechanism remained to be resolved.The IP method was used to screen interaction molecules of NgAgo protein and the homologous recombinase recA was identified as its interaction protein by mass spectrometry in this thesis.Then,the forward and reverse Co-IP was used to verify the interaction between NgAgo and endogenous recA,and the interaction was not affected by dealing with DNase I.By analyzing the interaction between purified recombinant NgAgo protein and recA protein,it was found that the two proteins could directly interact with each other.Further application of SPR technology revealed that the interaction affinity between NgAgo and recA was higher than 10 n M,suggesting that recA was the interaction protein of NgAgo.During homologous recombination,recA combined with single-stranded DNA and mediated the strand exchange between single-stranded DNA and homologous DNA to achieve homologous recombination.In this study,it was found that NgAgo protein did not significantly increase the binding of recA protein to single-stranded DNA,but significantly enhanced the recA-mediated homologous DNA strand exchange ability.The results showed that the mechanism of NgAgo mediated bacterial gene knockout was the combination of NgAgo protein and homologous recombinase recA,which enhanced the homologous DNA chain exchange ability mediated by recA,thus improving the efficiency of homologous recombination and achieved bacterial genome editing.4)PIWI domain was the main domain of NgAgo protein-mediated bacterial genome editing.NgAgo protein is mainly composed of four domains,including N-terminal,PAZ,MID and PIWI domains.In order to analyze the main domain of NgAgo-mediated bacterial genome editing,NgAgo was segmented into NgAgo-A(N-terminal),B(PAZ-MID-PIWI domain),C(N-PAZ domain)and D(MID-PIWI domain).It was found that NgAgo-A and NgAgo-C could not,while NgAgo-B and NgAgo-D could interact with recA and effectively mediated the lyi gene knockout of avian Pasteurella multocida.This indicated that the C-terminus of NgAgo protein was the main domain that mediates bacterial genome editing.In order to exclude the DNase activity of the PIWI domain,we mutated the enzyme active site of NgAgo-D protein.It was found that NgAgo D-M could still interact with recA,enhanced the homologous DNA strand exchange ability mediated by recA,and improved the homologous recombination efficiency to mediate the gene knockout of P.multocida and E.coli.It indicated that the C-terminus of NgAgo protein was the main domain that mediated bacterial genome editing and did not depend on its own DNase activity.In order to further analyze the main domain of NgAgo-mediated bacterial genome editing was MID or PIWI,so NgAgo was segmented into NgAgo-E(MID)and NgAgo-F(PIWI),found that PIWI was the main domain that interacted with recA and effectively mediated opa gene knockout in avian Pasteurella multocida.It indicated that the PIWI domain was the main domain of NgAgo protein-mediated bacterial genome editing and did not depend on its own DNase activity.5)Multiple pAgo proteins could efficiently mediate bacterial gene knockout and did not depend on its own DNase activity.The NgAgo protein could interact with recA through the PIWI domain and enhanced the efficiency of recA-mediated homologous recombination to mediate bacterial genome editing.This thesis further analyzed the functional conservation of the PIWI domain of other prokaryotic Ago proteins.Analyzed with Thermus thermophilus Argonaute(Tt Ago),Aquifex aeolicus Argonaute(Aa Ago)and Pyrococcus furiosus Argonaute(Pf Ago),found that all three pAgo(MID-PIWI regions)proteins could interact with the bacterial recA protein,which could efficiently mediate the knockout of the lyi and opa genes of P.multocida and did not depend on the DNase activity of PIWI.It indicated that the PIWI domains of Ago proteins of different prokaryotes were functionally conserved and could mediate bacterial genome editing.In conclusion,this study found that the pAgo protein with PIWI domain can mediate the genome editing of P.multocida and E.coli.The mechanism was not dependent on the hydrolase activity of gDNA targeted cleavage of genomic DNA,but the interaction of the PIWI domain of pAgo and recA to enhance the ability of recA-mediated homologous DNA chain exchange,thus improving the efficiency of homologous recombination.PAgo had the potential to be universal,simple,efficient and seamless tool for bacterial genome editing,which laid the foundation for studying on the pathogenic mechanism and prevention and control products of important pathogens.