Construction of srtA-Knockout Strain in Staphylococcus aureus by CRISPR/Cas9 Technology

doi:10.13560/j.cnki.biotech.bull.1985.2020-0195

Abstract

Abstract: This work aims to construct a srtA-deleted strain and srtA-complemented strain that is resistant to methicillin-resistant Staphylococcus aureus（MRSA）USA300 strain by CRISPR/Cas9,and to analyze its toxic effect on strain. The pCasSA-sgRNA plasmid was constructed using pCasSA vector and products of 3 pairs of srtA gRNAs. Then the pCasSA-sgRNA plasmid was modified through deletion S. aureus RN4220 strain,and transferred into USA300 strain,and the efficiency of cutting pCasSA-sgRNA plasmid was measured. The homologous sequences in the upstream（srtA-L）and downstream（srtA-R）of the srtA were amplified by PCR respectively,and their products were ligated by overlapping PCR. Then ligated products were inserted into pCasSA-sgRNA plasmid for constructing deletion pCasSA-sgRNA-srtA plasmid. The pCasSA-sgRNA-srtA plasmid was transferred into USA300 strain,and the srtA-deleted strains were obtained by screening. The growths of USA300 after the srtA deletion were compared,and their pathogenicity in mice were evaluated based on the survival rate of infected mice,bacteria burdens in organs,and histopathological changes in kidney. Meanwhile,the pLI50 plasmid was used as the vector to construct the complemented plasmid pLI50-srtA and complemented strain to verify whether the phenotypes were various in different strains. Two srtA-deleted strains were obtained after chloramphenicol screening. The deletion of srtA did not affect the growth of USA300;however,it reduced the mortality of the infected mice,as well as the bacteria burdens and pathological changes in kidney,which could be recovered by srtA complemented strains. The srtA-deleted strains and -complemented strains in the USA300 were successfully constructed in this study.

Key words: CRISPR/Cas9, Staphylococcus aureus, gene srtA, gene knockout, genetic complementation

JIANG Cheng-hui, ZENG Qiao-ying, WANG Meng, PAN Yang-yang, LIU Xu-ming, SHANG Tian-tian. Construction of srtA-Knockout Strain in Staphylococcus aureus by CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2020, 36(9): 253-265.

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