CRISPR/Cas9构建srtA基因敲除的金黄色葡萄球菌

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

摘要/Abstract

摘要： 利用CRISPR/Cas9技术构建耐甲氧西林金黄色葡萄球菌USA300的srtA基因缺失株及回补株,分析其对菌株毒力的影响。设计3对srtA基因sgRNA,以pCasSA为载体,构建pCasSA-sgRNA质粒。经缺陷型金黄色葡萄球菌RN4220菌株修饰后,转入USA300中,检测pCasSA-sgRNA质粒的切割效率。扩增并融合srtA基因左右同源臂并将其插入pCasSA-sgRNA质粒中,构建敲除质粒pCasSA-sgRNA-srtA。敲除质粒经修饰,转入USA300中,筛选并鉴定获得srtA基因缺失株。比较分析srtA基因缺失后对USA300菌株生长,小鼠存活率、器官载菌量及其肾脏组织病理学变化差异。同时,以pLI50质粒为载体,构建回补质粒pLI50-srtA及回补株,验证不同菌株表型是否存在差异。经氯霉素筛选,获得2个基因缺失株。与野生型菌株相比,srtA基因缺失后不影响USA300菌株的生长,但显著降低感染小鼠的死亡率,心脏、肾脏载菌量及肾脏组织病变程度。经srtA基因回补后其功能得以恢复。成功建立耐甲氧西林金黄色葡萄球菌USA300菌株srtA基因缺失株和回补株基因编辑方法。

关键词: CRISPR/Cas9, 金黄色葡萄球菌, srtA基因, 基因敲除, 基因回补

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

蒋成辉, 曾巧英, 王萌, 潘阳阳, 刘旭明, 尚天甜. CRISPR/Cas9构建srtA基因敲除的金黄色葡萄球菌[J]. 生物技术通报, 2020, 36(9): 253-265.

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.

参考文献

[1] Lowy FD.Staphylococcus aureus infections[J]. New England Journal of Medicine, 1998, 339(8):520-532.
[2] Smyth DS, Kafer JM, Wasserman GA, et al.Nasal carriage as a source of Staphylococcus aureus bacteremia[J]. New England Journal of Medicine, 2001, 344(1):11-16.
[3] Hou X, Wang M, Wen Y, et al.Quinone skeleton as a new class of irreversible inhibitors against Staphylococcus aureus sortase A[J]. Bioorg Med Chem Lett, 2018, 28(10):1864-1869.
[4] Wardenburg JB, Patel RJ, Schneewind O.Surface proteins and exotoxins are required for the pathogenesis of Staphylococcus aureus pneumonia[J]. Infect Immun, 2007, 75(2):1040-1044.
[5] 陈福广. 分选酶A在金黄色葡萄球菌引起的乳腺炎、菌血症和肺炎致病过程中的作用[D]. 长春:吉林大学, 2014.
Chen FG.Role of sorter A in the pathogenesis of mastitis, bacteremia and pneumonia caused by Staphylococcus aureus[D]. Changchun:Jilin University, 2014.
[6] Cossart P, Jonquieres R.Sortase, a universal target for therapeutic agents against Gram-positive bacteria[J]. Proc Natl Acad Sci USA, 2000, 97(10):5013-5015.
[7] Bowater R, Doherty AJ.Making ends meet:repairing breaks in bacterial DNA by non-homologous end-joing[J]. PLoS Genetics, 2006, 2(2):93-99.
[8] Spoto M, Guan C, Fleming E, et al.A universal, genomewide guidefinder for CRISPR/Cas9 targeting in microbial genomes[J]. mSphere, 2020, 5(1):10-20.
[9] Mougiakos I, Bosma EF, de Vos WM, et al. Next generation prokaryotic engineering:the CRISPR-Cas toolkit[J]. Trends in Biotechnology, 2016, 34(7):575-587.
[10] Gasiunas G, Barrangou R, Horvath P, et al.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. PNAS, 2012, 109(39):2579-2586.
[11] Xia J, Wang L, Zhu JB, et al.Expression of She-wanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering[J]. Biotechnology Letters, 2016, 38(1):117-122.
[12] Hong KQ, Liu DY, Chen T, et al.Recent advances in CRISPR/Cas9 mediated genome editing in Bacillus subtilis[J]. World Journal of Microbiology and Biotechnology, 2018, 34(10):151-155.
[13] 邢述永, 路志群, 夏海洋, 等. 构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统[J]. 微生物学通报, 2018, 45(12):2738-2750.
Xing SY, Lu ZQ, Xia HY, et al.CRISPR-Cas9-assisting efficient and sequential genome mutants in Mycobacterium smegmatis[J]. Microbiology China. 2018, 45(12):2738-2750.
[14] Jiang W, Bikard D, Cox D, et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 2016, 31(3):233-239.
[15] Oh JH, Van Pijkeren JP.CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri[J]. Nucleic Acids Res, 2014, 15(17):17-21.
[16] Michael EP, Mark RB, et al.Harnessing heterologous and endoge-nous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium[J]. Sci Rep, 2016, 6(5):125-129.
[17] Cobb RE, Wang J, Zhao HM.High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system[J]. ACS Synthetic Biology, 2015, 4(6):723-728.
[18] Khanzadi MN, Khan AA.CRISPR/Cas9:Nature’s gift to prokaryotes and an auspicious tool in genome editing[J]. Journal of Bsaic Microbiology, 2019, 10(6):1002-1005.
[19] Zhao C, Shu X, Sun B.Construction of a dCas9-bsaed gene knockdown system in Staphylococcus aureus[J]. Applied and Environmental Microbiology, 2017, 83(12):12-17.
[20] Chen W, Zhang Y, Yeo WS, et al.Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/Cas9 system[J]. Journal of the American Chemical Society, 2017, 139(10):3790-3795.
[21] Penewit K, Holmes EA, McLean K, et al. Efficient and scalable precision genome editing in Staphylococcus aureus through conditional recombineering and CRISPR/Cas9-mediated counterselection[J]. Mbio, 2018, 9(5):7-18.
[22] 张晓静, 冯世源, 杜崇涛, 等. 金黄色葡萄球菌OatA基因敲除菌株及其回补菌株的构建[J]. 现代生物医学进展, 2015, 4(15):25-29.
Zheng XJ, Feng SY, Du CT, et al.Construction of OatA mutant mutant and complementation of Staphylococcus aureus[J]. Progress in Modern Biomedicine, 2015, 4(15):25-29.
[23] Horvath PM, Kavalali ET, Monteggia LM.CRISPR/Cas9 system-mediated impairment of synaptobrevin/VAMP function in postmitotic hippocampal neurons[J]. Journal of Neuroscience Methods, 2016, 278(15):57-64.
[24] 郑小梅, 张晓立, 于建东, 等. CRISPR-Cas9介导的基因组编辑技术的研究进展[J]. 生物技术进展, 2015, 11(1):1-9.
Zheng XM, Zhang XL, Yu JD, et al.Advance in and application of CRISPR-Cas9 technology in bacteria[J]. Current Biotechnology, 2015, 11(1):1-9.
[25] Liu Y, Shi D, et al.Dracorhodin Perochlorate attenuates Staphylo-coccus aureus USA300 virulence by decreasing α-toxin expression[J]. World J Microbiol Biotechnol, 2017, 33(1):117-121.
[26] Hornak JP, Anjum S, Reynoso D, et al.Adjunctive ceftaroline in combination with daptomycin or vancomycin for complicated methicillin-resistant bacteremia after monotherapy failure[J]. Ther Adv Infect Dis, 2019, 10(6):117-128.
[27] Martens, Evan, Demain, et al. The antibiotic resistance crisis, with a focus on the United States[J]. The Journal of Antibiotics:An International Journal, 2017, 70(5):1030-1039.
[28] 张奇文, 凌晨, 吴学林, 等. 单增李斯特菌srtA基因缺失株的构建及srtA基因对其毒力的影响研究[J]. 中国动物传染病学报, 2019, 27(4):23-31.
Zhang QW, Ling C, Wu XL, et al.Construction and virulence evaluation of the srtA gene deletion strain of Listeria monocytogenes[J]. Chinese Journal of Animal Infectious Diseases, 2019, 27(4):23-31.
[29] 张静玲, 李国明, 邱景富. 金黄色葡萄球菌生物膜形成能力及其耐甲氧西林相关性[J]. 中国消毒学杂志, 2018. 35(1):14-16.
Zhang JL, Li GM, Qiu JF.Research on relationship between methicillin resistance of Staphylococcus aureus clinical isolates and its biofilm forming ability[J]. Chinese Journal of Antisepsis, 2018. 35(1):14-16.