Application Of CRISPR System In Vibrio Parahaemolyticus

Clustered regularly interspaced short palindromic repeats(CRISPR)have been recently identified in most archaea and many bacteria.The system which composed of CRISPR and CRISPR-associated genes can effectively defend against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids,limiting horizontal gene transfer.Also,the function of participating in the regulation of bacterial virulence has been found in the CRISPR system of foodborne pathogens.There are differences in the diversity of CRISPR in foodborne pathogens among different strains,which provides a more reliable foundation for the genetic evolution and molecular typing of foodborne pathogens.Because of its unique mechanism,the CRISPR system become a genomic editing tool which is widely used in prokaryotes and eukaryotes.The application of CRISPR system provides an efficient and accurate tool for studying functional gene and interaction among genes in future.In this study,these two functions of CRISPR used in Vibrio parahaemolyticus.The molecular typing and evolution of Vibrio parahaemolyticus were analyzed by detecting CRISPR loci and the gene editing was carried out by CRISPR / Cas9 technique for the first time to construct a new gene editing tool for V.parahaemolyticus.1.Detection and analysis of CRISPR in V.parahaemolyticusIn this chapter,the CRISPR loci in 79 strains of Vibrio parahaemolyticus(VP)were examined,and its structural diversities in different strains were analyzed.The primers of convincing CRISPR structure CRISPR-1 were designed by using the sequence according to CRISPR database,while the primers of questionable CRISPR structure CRISPR-2 were designed according to the literature.The PCR approach was used to detect the CRISPR locus in all 79 strains,and all CRISPR sequences were analyzed using CRISPR Finder.Furthermore,the structural diversities of CRISPR in different sources of VP were analyzed using bioinformatics methods.The results found that the positive rate of CRISPR-1 and CRISPR-2 was 92.41%,96.20% respectively.The strains possessed with these two locus accounted for 89.87% of the total strains,and only one strain did not contain any locus.There was no difference between the repeats of CRISPR-1 and CRISPR-2 from different sources of VP,while,the spacers of those two locus in clinical strains had more variations than those in environmental strains.For this reason,two CRISPR locus formed 8 spectral patterns(numbers A-H)according to the variations of spacers.Except type F,the patterns A-E,G only were found in the clinical strains.And the type H which contains none of locus was only found in environmental strains.Therefore,CRISPR commonly existed in VP and the structures of CRISPR between environmental and clinical strains were differences.2.Application of CRISPR / Cas9 technique in Escherichia coliAs an emerging genome modification method which is widely used in the genomic editing of prokaryotes and eukaryotes,the CRISPR/Cas9 can edit genome efficiently and accurately.In this study,the CRISPR-based method was used successfully for single allelic exchange in E.coli.A binary vector was constructed with CRISPR/Cas9 and ?-Red recombinase.Donor genes were then knockout through overlapping PCR.The recombinant vector plasmid was subsequently electrotransformed into E.coli MG1655 to edit the qseB gene associated with cell motility.The phenotype and sequencing were used to confirm whether the gene was knocked out successfully.The results suggested that absence of the qseB gene increases cell motility.The CRISPR/Cas9 and ?-Red combination technic further improved the efficient of gene modification and shortened the period of experiment.3.Application of CRISPR / Cas9 technique in VPThe CRISPR/Cas9 technique are used to introduce precise mutations in the genome of VP.The deletion of two key genes(aphA,opaR)of Quorum sensing(QS)are used to verify the feasibility of the experiment.The pTarget series have two versions,pTarget T and pTargetF,which have donor DNA for recombination supplied in the plasmid pTarget and not supplied,respectively.The recombinant vector plasmid was subsequently electrotransformed into VP31 competent cells to edit the aphA and opaR gene associated with biofilm formation.As a result,transformants containing double plasmids were not obtained,however,only transformants with one of the plasmids were obtained in VP31,indicating that the plasmid was incompatible in VP.In order to solve this technical problem,the indigenous plasmids of VP will be cured by experiments and transformated the existing plasmids in order to realize the application of CRISPR/Cas9 technology in VP.The research of this subject provides a basis for exploring the function of VP's own CRISPR / Cas system and constructing a new gene editing tool for VP.