CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus

Abstract

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.

Keywords: Staphylococci; gene; genotyping; molecular genetics; plasmid.