CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus  

作　　者：Qi Liu Yu Jiang Lei Shao Ping Yang Bingbing Sun Sheng Yang Daijie Chen 

机构地区：[1]School of Pharmacy, Fudan University, Shanghai 201203, China [2]Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China [3]Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China [4]Shanghai Research and Development Center of Industrial Biotechnology, Shanghai 201206, China [5]School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China

出　　处：《Acta Biochimica et Biophysica Sinica》2017年第9期764-770,共7页生物化学与生物物理学报（英文版）

摘　　要：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 Pxy/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.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 Pxy/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.

关 键 词：STAPHYLOCOCCI PLASMID molecular genetics GENOTYPING GENE 

分 类 号：Q78[生物学—分子生物学] TS207.4[轻工技术与工程—食品科学]