Analysis Of The Gene Structure And Establishment Of Detection Method Of CRISPR-Cas System In Campylobacter Jejuni

The CRISPR-Cas system is widely distributed in bacteria and archaea,with 34%of bacteria and 61%of archaea genomes containing the CRISPR-Cas system.In recent years,the CRISPR-Cas system has been shown to have immune defense functions,acquiring short nucleic acid sequences from invasive mobile genetic elements(MGEs).The gene structure of the CRISPR-Cas system is complex,with multiple Cas protein families,and many microbial strains lack partial or complete gene structures of the CRISPR-Cas system.Understanding the diversity of CRISPR-Cas system gene structures in the same species,through evolutionary analysis,can provide insight into why species choose to acquire or lose the CRISPR-Cas system in evolution,and also provide ideas for exploring other biological mechanisms.The exact pathogenesis of Campylobacter jejuni(C.jejuni)in the development of clinical diseases is largely unknown.Unlike other intestinal pathogens,C.jejuni does not contain toxins that clearly cause disease and most cases of C.jejuni infections are selflimiting,making research into the pathogenesis of C.jejuni challenging.C.jejuni has been reported to have only one CRISPR-Cas system,but research on the gene structure of the CRISPR-Cas system in C.jejuni is in its early stages.The complete structure of the CRISPR-Cas system includes the cas gene cluster,noncoding CRISPR loci,trans-activating RNA(tracrRNA),and leader sequences.In order to explore the specific gene structure features of the CRISPR-Cas system in C.jejuni,this study analyzed the coding and non-coding genes that make up the CRISPR-Cas system for the first time using comparative genomic methods based on the whole genome sequences of 486 laboratory strains and 1318 foreign strains with different backgrounds.The study also analyzed the genomic composition and conservation level of different CRISPR-Cas system structures in C.jejuni.The research results can be summarized as follows:1.Gene structure analysis and establishment of classification methodBy aligning the coding and non-coding genes that make up the complete CRISPR-Cas system in the standard strain NCTC 11168 with all bacterial samples,and based on the different combinations of these genes in C.jejuni,as well as the composition of CRISPR loci,this study for the first time distinguished the gene structures of complete,incomplete,and completely absent CRISPR-Cas systems.The three types of C.jejuni CRISPR-Cas system structures were defined as type Ⅱ-C for the complete system,type 3C for the system lacking CRISPR loci,and type No for the complete absence of CRISPR-Cas system structure.2.Comparison of multiple detection methods and establishment of a detection methodThree different methods for detecting CRISPR-Cas systems were used,using bioinformatics software and PCR methods to analyze the samples,and the accuracy of different detection methods was compared.Finally,a detection method using BLASTn technology to detect the C jejuni CRISPR-Cas system was established based on the gene structure of the three types of CRISPR-Cas systems present in C.jejuni.By comparing the conserved cas gene cluster of the Ⅱ-C type system and the two conserved non-coding sequences flanking the CRISPR locus in C jejuni,the structure components of the CRISPRCas system in the strain were identified.If the cas gene cluster is complete and the length of the sequence between the two conserved non-coding sequences is greater than 96 bp,indicating that there are at least two repeat sequences,then the strain has a complete Ⅱ-C type CRISPR-Cas system;if the cas gene cluster is complete but there is only one repeat sequence,then the strain has a 3C type CRISPR-Cas system lacking the CRISPR locus;if all gene structures are missing,then the strain has a No type CRISPR-Cas system.3.Comparative analysis of CRISPR-Cas systems in domestic and foreign strainsFinally,in order to validate the applicability and accuracy of the established analysis and detection methods,the BLASTn detection method was used to analyze the CRISPR-Cas system gene structure types of 1318 foreign C.jejuni strains.The distribution differences of the three types of CRISPR-Cas systems in domestic and foreign C.jejuni strains were small,with differences in the distribution rates of the Ⅱ-C and No types within 10%,In summary,this study analyzed all possible situations of the CRISPR-Cas system in C.jejuni,covering discussions on the cas gene cluster,CRISPR locus,and other related key genes.It established a standardized analysis method for studying the gene structure of the CRISPR-Cas system in C.jejuni and provides a theoretical basis for further exploring the significance of the CRISPR-Cas system in C.jejuni.