CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus

Qi Liu, Yu Jiang, Lei Shao, Ping Yang, Bingbing Sun, Sheng Yang, and Daijie Chen

School of Pharmacy, Fudan University, Shanghai 201203, China & Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China & Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

Acta Biochim Biophys Sin 2017, 49: 764–770; doi: 10.1093/abbs/gmx074

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 P-xyl/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.

CRISPR-Cas9-mediated gene editing targeting at the tgt gene in the Staphylococcus aureus RN4220 genome

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