Molecular Mechanism Of Genomic Remodeling Mediated By Type ?-A CRISPR-Cas System In Staphylococcus Aureus

Staphylococcus aureus is a bacterial pathogen that can cause many infectious diseases and serious public health threat in human.The overuse of antibiotics results in the emergence and prevalence of drug-resistant strains.Methicillin-resistant Staphylococcus aureus(MRSA)can survive in the existence of ?-lactam antibiotics and causes persistent infection.The resistance to ?-lactam antibiotics among MRSA strains is due to the acquisition of the resistance gene mecA,which is carried on the mobile genetic element SCCmec and encodes an additional penicillin-binding protein PBP2a with low affinity for ?-lactam antibiotics.The SCCmec can conduct horizontal transfer among staphylococcal strains and accordingly lead to the prevalence of methicillin resistance.Clustered regularly interspaced short palindromic repeats(CRISPRs)and CRISPR associated proteins(Cas)constitute an adaptive immunity system that protects archaea and bacteria from threats of foreign nucleic acids.According to the constitution and function of Cas proteins,CRISPR-Cas systems are currently classified into 5 distinctive types and diverse subtypes.Recent studies have mainly focused on the molecular mechanism of immunity function and relevant application in gene editing.However,a few studies reported that CRISPR-Cas systems can modulate genome stability.A recent study has reported that among 4,500 spacers from various organisms,35%show homologs to chromosomal sequence in the NCBI database.Some of them target genes within integrated MGEs.This result implies that the occurrence of chromosomal-targeting spacer is not an accident,which may play an important role in bacterial evolution.Our research elucidated that chromosomal targeting by the type ?-A CRISPR-Cas system toward the gene mecA within SCCmec can influence the genome stability and drug resistance of clinical MRSA strain.We constructed artificial CRISPR plasmids with the sequence in the coding strand of mecA as the protospacer,and then transformed plasmids into the clinical MRSA strain AH1.By genomic sequencing,we found that CRISPR-mediated chromosomal targeting is lethal to transformants.However,a minority of bacteria can avoid the CRISPR attack by genomic remodeling.Most of escapers deleted the large-scale fragments(?16 kb)including the target sequence within SCCmec.The remaining escapers survived due to the deletion of the anti-mecA spacers or loss-of-function mutations in cas genes required for targeting.The transcription efficiencies of different length leader sequences were detected by real-time quantitative reverse transcription-PCR.The naive 252 bp leader sequence exhibited obvious transcriptional activity.As a result,the 252 bp leader was chosen as the promoter of the artificial CRISPR array in our research.In addition,we found that a single spacer is sufficient for targeting.We also demonstrated that chromosomal targeting by the type ?-A CRISPR-Cas system is transcription-dependent.CRISPR plasmids with spacers targeting the coding strand of mecA showed obvious targeting capacity.We changed the size of spacers in artificial CRISPR arrays,and the results indicated that maturation of crRNAs is independent of the sequence and size of spacers.All primary CRISPR transcripts were processed into mature crRNAs with the same sizes of 43 and 37 nt.Nevertheless,spacer size played an important role in CRISPR targeting.The targeting efficiency of the CRISPR-Cas system is associated with the complementarity between crRNAs and protospacers,and 10 to 13 nucleotides truncation of spacers partially blocks CRISPR attack and more than 13 nucleotides truncation can fully abolish targeting.Interestingly,avoiding base pairings in the upstream region of protospacers is also necessary for CRISPR targeting.Successive trinucleotide complementarity between the 5,tag of crRNAs and protospacers at position-1 to-5 can disrupt CRISPR targeting.Our study proves that chromosomal targeting by CRISPR-Cas systems can promote bacterial genome remodeling and further elucidated the molecular mechanism of type ?-A CRISPR-Cas systems.These findings may provide novel insight into clinical applications of CRISPR-Cas systems in the treatment of MRS A infection.