Analyzing The CRISPR Structures In Staphylococcus Aureus And Using CRISPR/Cas9 Technic To Target The MecA Gene

Objectives1.Analyzing the structural features and the repeat sequences’ RNA secondary structure stability of the 45 identified staphylococcus aureus CRISPR loci in the CRISPR database.Exploring its functional characteristics and its potential functional influencing factors.2.Analyzing the homology between spacer sequences and the sequences of foreign plasmids or phages,as well as the potential association between the CRISPR types and the MLST types,so that we can explain the relationship between the CRISPR loci and the genetic evolution of strains.3.By using the CRISPR/Cas9 system in the Escherichia Coli to target the mecA and to eliminate the plasmids located in,we can explore the CRISPR/Cas9 system’s function of knocking out a certain drug-resistant gene specifically and provide us a new possible method of finding a new kind of antibiotics.Methods1.The general information about the CRISPR structures was searched from the CRISPR database(http://crispr.u-psud.fr/).The Clustal X was used to perform multi-sequences alignment and the Tree View 1.6.6 was used to draw the unrooted tree plots.The RNA secondary structures were predicted by RNA fold Web Server(http://rna.tbi.univie.ac.at/).2.NCBI BLAST,BLASTP and PUBMED database were used to search the similar sequences of the DNA and Cas proteins as well as the information of MLST types of the strains involved.3.The mecA gene was amplified by PCR.The pET-21a(+)-mecA plasmid was constructed by gel extraction of mecA gene,connecting of the mecA gene to the T-vector,enzyme digestion and connecting of the mecA gene to the plasmid pET-21a(+).Besides,the result was validated by DNA sequencing.4.The oligos were designed by software sgRNAcas9 v2.0 and then connected to the plasmid pCas9,giving plasmid pCas9::mecA.5.The differences of the RNA secondary structure’s stability were analyzed by SPSS21.0 and the Cas9 protein’ function of eliminating the plasmid containing mecA gene were also proved by the SPSS21.0 software.Results1.In the database,there were 45 identified CRISPR loci contained in the 32 staphylococcus aureus and among them,19 strains each had one identified locus in the genome,counting for 59.4%of the total strains.Besides,the remaining strains each had two CRISPR loci in their genome.The 45 repeat sequences could all form conserved dumbbell shaped RNA secondary structures with two "rings"located at two opposite ends and one "stem" in the middle.According to the results of multi-sequences alignment,the repeat sequences could be divided into 15 groups which could be further classified into 3 clusters.In the first cluster,the RNA secondary structures with 5 base pairs in the "stem" had lower MFE(minimum free energy)than those RNA secondary structures with "stems" of 3 base pairs s long.In the second cluster,the structures with "longer stems" had lower MFE.In the third cluster,the structures all had "stems" of equal length,but the sequence with higher "GC" contend had a lower MFE.2.There were 5 spacer sequences in strain 08BA02176 and strain MSHR1132,which could find homologous sequences from the foreign plasmids or phages.Among them one spacer showed high similarity with a foreign plasmid that could encode the Panton-Valentine leukocidin.3.The CRISPR loci in the database belonged to 17 types and 3 clusters.In the first cluster,at least 75.0%of the strains that had the same CRISPR types belonged to the same MLST types.In the second cluster,2 strains of CRISPR type "A" all belonged to MLST type 398.In the last cluster,the 2 strains of different CRISPR types didn’t have the same MLST types either.CRISPR loci sharing the same CRISPR types always had similar even identical adjacent proteins encoding loci(≥3).4.Plasmid pET-21a(+)-mecA and plasmid pCas9::mecA were finally successfully constructed.In the bacteria Escherichia Coli BL21(D3),the plasmid pCas9::mecA could successfully eliminate the plasmid pET-2 or inhibit its replication violently.Whereas,the control plasmid pCas9 didn’t show this effect.At the same time neither the pCas9::mecA nor pCas9 could eliminate the plasmid pET-21a(+),a plasmid that don’t contain the mecA gene.Conclusions1.The number of identified CRISPR loci in the genome of staphylococcus aureus is relatively small.The CRISPR loci whose repeat sequences’ RNA secondary structures have more "stem" base pairs and higher "GC" contents tend to be more likely to bear a function.2.CRISPR loci can move among different but genetically related strains.The CRISPR loci in strain 08BA02176 and MSHR1132 may be inherited from one ancestor or be gained from the S.lugdunensis species by horizontal gene transfer.The same type of MLST strains may show higher affinity to the same type of CRISPR loci,as well as the adjacent conserved proteins that may bear some important functions.3.pCas9::mecA may eliminate the plasmid pET-21a(+)-mecA by targeting the mecA gene.Thus it can limt the transmission of mecA among staphylococcus aureus and protect the sensitive isolates from becoming the MRSA.