金黄色葡萄球菌基因组编辑技术
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摘要
金黄色葡萄球菌是一种革兰氏阳性病原菌,可引发多种严重感染。分子遗传学操作是研究金黄色葡萄球菌基因功能和分子感染机制的重要手段。金黄色葡萄球菌因其限制性修饰系统使得对其进行遗传学操作总显得费时费力。此外,较低的DNA转化效率和同源重组率也一直制约着相关研究的开展。本研究列举了近年来主流的金黄色葡萄球菌基因组编辑方法,包括基于基因等位替换的抗生素筛选和反向筛选系统、基于Ⅱ型内含子剪切的TargeTron系统以及重组的CRISPR/Cas9系统,从技术层面较为详细地介绍和比较了上述编辑技术的操作原理和试验流程,以期为相关领域的研究人员提供技术参考。
Staphylococcus aureus is a Gram-positive pathogen that causes a variety of serious infections. Molecular genetic manipulation is an important tool for understanding genes function and molecular mechanism of S. aureus infection. Genetic manipulation in S. aureus is always time consuming and labor intensive due to its restrictionmodification system. Besides, poor efficiency in DNA transformation and homologous recombination have also restricted the development of related research. In this study, the main genome editing methods of S. aureus in recent year were listed, including antibiotic screening and inverse screening systems based on allelic substitution, TargeTron system based on group Ⅱ intron splicing and recombinant CRISPR/Cas9 system. The operating principles and experiment procedures of these editing techniques were introduced in detail and compared at technical level, in order to provide technical reference for researchers dedicated in the S. aureus research fields.
Staphylococcus aureus is a Gram-positive pathogen that causes a variety of serious infections. Molecular genetic manipulation is an important tool for understanding genes function and molecular mechanism of S. aureus infection. Genetic manipulation in S. aureus is always time consuming and labor intensive due to its restrictionmodification system. Besides, poor efficiency in DNA transformation and homologous recombination have also restricted the development of related research. In this study, the main genome editing methods of S. aureus in recent year were listed, including antibiotic screening and inverse screening systems based on allelic substitution, TargeTron system based on group Ⅱ intron splicing and recombinant CRISPR/Cas9 system. The operating principles and experiment procedures of these editing techniques were introduced in detail and compared at technical level, in order to provide technical reference for researchers dedicated in the S. aureus research fields.
引文
Archer G.L.,1998,Staphylococcus aureus:a well-armed pathogen,Clinical Infectious Diseases,26(5):1179-1181
Arnaud M.,Chastanet A.,and DébarbouilléM.,2004,New vector for efficient allelic replacement in naturally nontransformable,low-GC-content,gram-positive bacteria,Appl Environ.Microbiol.,70(11):6887-6891
Bae T.,and Schneewind O.,2006,Allelic replacement in Staphylococcus aureus with inducible counter-selection,Plasmid55(1):58-63
Brückner R.,1997,Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus,FEMS Microbiology Letters151(1):1-8
Chambers H.F.,1997,Methicillin resistance in staphylococci molecular and biochemical basis and clinical implications Clin.Microbiol.Rev.,10(4):781-791
Chen W.Z.,Zhang Y.F.,Yeo W.S.,Bae T.,and Ji Q.J.,2017Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/Cas9 system,J.Am Chem.Soc.,139(10):3790-3795
DeLeo F.R.,and Chambers H.F.,2009,Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era,The Journal of Clinical Investigation,119(9):2464-2474
Doudna J.A.,and Charpentier E.,2014,The new frontier of genome engineering with CRISPR-Cas9,Science,346(6213)1258096
Gardam M.A.,2000,Is methicillin-resistant Staphylococcus aureus an emerging community pathogen,A review of the literature,The Canadian Journal of Infectious Diseases,11(4)202-211
Gordon R.J.,and Lowy F.D.,2008,Pathogenesis of methicillin-resistant Staphylococcus aureus infection,Clinical Infectious Diseases,46(Suppl.5):S350-S359
Karberg M.,Guo H.,Zhong J.,Coon R.,Perutka J.,and Lambowitz A.M.,2001,GroupⅡintrons as controllable gene targeting vectors for genetic manipulation of bacteria,Nature Biotechnology,19(12):1162-1167
Kato F.,and Sugai M.,2011,A simple method of markerless gene deletion in Staphylococcus aureus,Journal of Microbiological Methods,87(1):76-81
Kiedrowski M.R.,Kavanaugh J.S.,Malone C.L.,Mootz J.M.,Voyich J.M.,Smeltzer M.S.,Bayles K.W.,and Horswill A.R.,2011,Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus,PLoS One,6(11):e26714
Kreiswirth B.N.,Lofdahl S.,Betley M.J.,O'Reilly M.,Schlievert P.M.,Bergdoll M.S.,and Novick R.P.,1983,The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage,Nature,305(5936):709-712
Leibig M.,Krismer B.,Kolb M.,Friede A.,Gotz F.,and Bertram R.,2008,Marker removal in staphylococci via Cre recombinase and different lox sites,Appl.Environ.Microbiol.,74(5):1316-1323
Lewis A.M.,Matzdorf S.S.,Endres J.L.,Windham I.H.,Bayles K.W.,and Rice K.C.,2015,Examination of the Staphylococcus aureus nitric oxide reductase(saNOR)reveals its contribution to modulating intracellular NO levels and cellular respiration,Molecular Microbiology,96(3):651-669
Mcdevitt D.,Wann E.R.,and Foster T.J.,1993,Recombination at the coagulase locus in Staphylococcus aureus:plasmid integration and amplification,Journal of General Microbiology,139(4):695-706
Nair D.,Memmi G.,Hernandez D.,Bard J.,Beaume M.,Gill S.,Francois P.,and Cheung A.L.,2011,Whole-genome sequencing of Staphylococcus aureus strain RN4220,a key laboratory strain used in virulence research,identifies mutations that affect not only virulence factors but also the fitness of the strain,Journal of Bacteriology,193(9):2332-2335
Novick R.P.,1991,Genetic systems in staphylococci,Methods in Enzymology,204:587-636
Roux A.,Todd D.A.,Velázquez J.V.,Cech N.B.,and Sonenshein A.L.,2014,CodY-mediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals,Journal of Bacteriology,196(6):1184-1196
Sahukhal G.S.,Batte J.L.,and Elasri M.O.,2015,msaABCRoperon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus,FEMSMicrobiol.Lett.,362(4):1-10
Singh M.,Matsuo M.,Sasaki T.,Morimoto Y.,Hishinuma T.,and Hiramatsu K.,2017,In vitro tolerance of drug-naive Staphylococcus aureus strain FDA209P to vancomycin,Antimicrobial Agents and Chemotherapy,61(2):e01154-16
Yao J.,Zhong J.,Fang Y.,Geisinger E.,Novick R.P.,and Lambowitz A.M.,2006,Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB groupⅡintron splicing,RNA,12(7):1271-1281
Zhang F.,Wen Y.,and Guo X.,2014,CRISPR/Cas9 for genome editing:progress,implications and challenges,Human Molecular Genetics,23(R1):R40-R46
Arnaud M.,Chastanet A.,and DébarbouilléM.,2004,New vector for efficient allelic replacement in naturally nontransformable,low-GC-content,gram-positive bacteria,Appl Environ.Microbiol.,70(11):6887-6891
Bae T.,and Schneewind O.,2006,Allelic replacement in Staphylococcus aureus with inducible counter-selection,Plasmid55(1):58-63
Brückner R.,1997,Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus,FEMS Microbiology Letters151(1):1-8
Chambers H.F.,1997,Methicillin resistance in staphylococci molecular and biochemical basis and clinical implications Clin.Microbiol.Rev.,10(4):781-791
Chen W.Z.,Zhang Y.F.,Yeo W.S.,Bae T.,and Ji Q.J.,2017Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/Cas9 system,J.Am Chem.Soc.,139(10):3790-3795
DeLeo F.R.,and Chambers H.F.,2009,Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era,The Journal of Clinical Investigation,119(9):2464-2474
Doudna J.A.,and Charpentier E.,2014,The new frontier of genome engineering with CRISPR-Cas9,Science,346(6213)1258096
Gardam M.A.,2000,Is methicillin-resistant Staphylococcus aureus an emerging community pathogen,A review of the literature,The Canadian Journal of Infectious Diseases,11(4)202-211
Gordon R.J.,and Lowy F.D.,2008,Pathogenesis of methicillin-resistant Staphylococcus aureus infection,Clinical Infectious Diseases,46(Suppl.5):S350-S359
Karberg M.,Guo H.,Zhong J.,Coon R.,Perutka J.,and Lambowitz A.M.,2001,GroupⅡintrons as controllable gene targeting vectors for genetic manipulation of bacteria,Nature Biotechnology,19(12):1162-1167
Kato F.,and Sugai M.,2011,A simple method of markerless gene deletion in Staphylococcus aureus,Journal of Microbiological Methods,87(1):76-81
Kiedrowski M.R.,Kavanaugh J.S.,Malone C.L.,Mootz J.M.,Voyich J.M.,Smeltzer M.S.,Bayles K.W.,and Horswill A.R.,2011,Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus,PLoS One,6(11):e26714
Kreiswirth B.N.,Lofdahl S.,Betley M.J.,O'Reilly M.,Schlievert P.M.,Bergdoll M.S.,and Novick R.P.,1983,The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage,Nature,305(5936):709-712
Leibig M.,Krismer B.,Kolb M.,Friede A.,Gotz F.,and Bertram R.,2008,Marker removal in staphylococci via Cre recombinase and different lox sites,Appl.Environ.Microbiol.,74(5):1316-1323
Lewis A.M.,Matzdorf S.S.,Endres J.L.,Windham I.H.,Bayles K.W.,and Rice K.C.,2015,Examination of the Staphylococcus aureus nitric oxide reductase(saNOR)reveals its contribution to modulating intracellular NO levels and cellular respiration,Molecular Microbiology,96(3):651-669
Mcdevitt D.,Wann E.R.,and Foster T.J.,1993,Recombination at the coagulase locus in Staphylococcus aureus:plasmid integration and amplification,Journal of General Microbiology,139(4):695-706
Nair D.,Memmi G.,Hernandez D.,Bard J.,Beaume M.,Gill S.,Francois P.,and Cheung A.L.,2011,Whole-genome sequencing of Staphylococcus aureus strain RN4220,a key laboratory strain used in virulence research,identifies mutations that affect not only virulence factors but also the fitness of the strain,Journal of Bacteriology,193(9):2332-2335
Novick R.P.,1991,Genetic systems in staphylococci,Methods in Enzymology,204:587-636
Roux A.,Todd D.A.,Velázquez J.V.,Cech N.B.,and Sonenshein A.L.,2014,CodY-mediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals,Journal of Bacteriology,196(6):1184-1196
Sahukhal G.S.,Batte J.L.,and Elasri M.O.,2015,msaABCRoperon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus,FEMSMicrobiol.Lett.,362(4):1-10
Singh M.,Matsuo M.,Sasaki T.,Morimoto Y.,Hishinuma T.,and Hiramatsu K.,2017,In vitro tolerance of drug-naive Staphylococcus aureus strain FDA209P to vancomycin,Antimicrobial Agents and Chemotherapy,61(2):e01154-16
Yao J.,Zhong J.,Fang Y.,Geisinger E.,Novick R.P.,and Lambowitz A.M.,2006,Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB groupⅡintron splicing,RNA,12(7):1271-1281
Zhang F.,Wen Y.,and Guo X.,2014,CRISPR/Cas9 for genome editing:progress,implications and challenges,Human Molecular Genetics,23(R1):R40-R46