Staphylococcus aureus is an important pathogenic bacterium prevalent in nosocomial infections and associated with high morbidity and mortality rates,which arise from the significant pathogenicity and multi-drug resistance.However,the typical genetic manipulation tools used to explore the relevant molecular mechanisms of S.aureus have multiple limitations:leaving a scar in the genome,comparatively low gene-editing efficiency,and prolonged experimental period.Here,we present a single-plasmid based on the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system which allows rapid and efficient chromosomal manipulation in S.aureus.The plasmid carries the cas9 gene under the control of the constitutive promoter Pxyl/tet,a single guide RNA-encoding sequence transcribed via a strong promoter Pspac,and donor DNA used to repair the double strand breaks.The function of the CRISPR/Cas9 vector was demonstrated by deleting the tgt gene and the rocA gene,and by inserting the erm R cassette in S.aureus.This research establishes a CRISPR/Cas9 genome editing tool in S.aureus,which enables marker-free,scarless and rapid genetic manipulation,thus accelerating the study of gene function in S.aureus.