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Recombineering in Staphylococcus aureus

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Recombineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2479))

Abstract

Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.

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References

  1. Klevens RM, Morrison MA, Nadle J et al (2007) Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA 298:1763–1771. https://doi.org/10.1001/jama.298.15.1763

    Article  CAS  PubMed  Google Scholar 

  2. Wertheim HFL, Vos MC, Ott A et al (2004) Risk and outcome of nosocomial Staphylococcus aureus bacteraemia in nasal carriers versus non-carriers. Lancet 364:703–705. https://doi.org/10.1016/S0140-6736(04)16897-9

    Article  PubMed  Google Scholar 

  3. Liu C, Bayer A, Cosgrove SE et al (2011) Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary. Clin Infect Dis 52:285–292. https://doi.org/10.1093/cid/cir034

    Article  PubMed  Google Scholar 

  4. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339:520–532. https://doi.org/10.1056/NEJM199808203390806

    Article  CAS  PubMed  Google Scholar 

  5. Prax M, Lee CY, Bertram R (2013) An update on the molecular genetics toolbox for staphylococci. Microbiology 159:421–435. https://doi.org/10.1099/mic.0.061705-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Monk IR, Shah IM, Xu M et al (2012) Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3. https://doi.org/10.1128/mBio.00277-11

  7. Liu Q, Jiang Y, Shao L et al (2017) CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus. Acta Biochim Biophys 49:764–770. https://doi.org/10.1093/abbs/gmx074

    Article  CAS  Google Scholar 

  8. Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98:6742–6746. https://doi.org/10.1073/pnas.121164898

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Datta S, Costantino N, Zhou X, Court DL (2008) Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proc Natl Acad Sci U S A 105:1626–1631. https://doi.org/10.1073/pnas.0709089105

    Article  PubMed  PubMed Central  Google Scholar 

  10. Barrangou R, van Pijkeren J-P (2016) Exploiting CRISPR-Cas immune systems for genome editing in bacteria. Curr Opin Biotechnol 37:61–68. https://doi.org/10.1016/j.copbio.2015.10.003

    Article  CAS  PubMed  Google Scholar 

  11. Reisch CR, Prather KLJ (2015) The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Sci Rep 5:15096. https://doi.org/10.1038/srep15096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223. https://doi.org/10.1038/nprot.2008.227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sawitzke JA, Thomason LC, Costantino N et al (2007) Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol 421:171–199. https://doi.org/10.1016/S0076-6879(06)21015-2

    Article  CAS  PubMed  Google Scholar 

  14. Nyerges Á, Csörgő B, Nagy I et al (2016) A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc Natl Acad Sci U S A 113:2502–2507. https://doi.org/10.1073/pnas.1520040113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Penewit K, Holmes EA, McLean K et al (2018) Efficient and scalable precision genome editing in Staphylococcus aureus through conditional recombineering and CRISPR/Cas9-mediated counter selection. mBio 9. https://doi.org/10.1128/mBio.00067-18

  16. Watanabe Y, Cui L, Katayama Y et al (2011) Impact of rpoB mutations on reduced vancomycin susceptibility in Staphylococcus aureus. J Clin Microbiol 49:2680–2684. https://doi.org/10.1128/JCM.02144-10

    Article  PubMed  PubMed Central  Google Scholar 

  17. Sun Z, Deng A, Hu T et al (2015) A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35. Appl Microbiol Biotechnol 99:5151–5162. https://doi.org/10.1007/s00253-015-6485-5

    Article  CAS  PubMed  Google Scholar 

  18. Van Pijkeren J-P, Neoh KM, Sirias D et al (2012) Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri. Bioengineered 3:209–217. https://doi.org/10.4161/bioe.21049

    Article  PubMed  PubMed Central  Google Scholar 

  19. Selle K, Barrangou R (2015) Harnessing CRISPR-Cas systems for bacterial genome editing. Trends Microbiol 23:225–232. https://doi.org/10.1016/j.tim.2015.01.008

    Article  CAS  PubMed  Google Scholar 

  20. Jiang W, Bikard D, Cox D et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. https://doi.org/10.1038/nbt.2508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Charpentier E, Anton AI, Barry P et al (2004) Novel cassette-based shuttle vector system for gram-positive bacteria. Appl Environ Microbiol 70:6076–6085. https://doi.org/10.1128/AEM.70.10.6076-6085.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sau S, Sun J, Lee CY (1997) Molecular characterization and transcriptional analysis of type 8 capsule genes in Staphylococcus aureus. J Bacteriol 179:1614–1621

    Article  CAS  Google Scholar 

  23. van der Vossen JM, van der Lelie D, Venema G (1987) Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol 53:2452–2457

    Article  Google Scholar 

  24. Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. https://doi.org/10.1016/j.cell.2013.02.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Monk IR, Tree JJ, Howden BP et al (2015) Complete bypass of restriction systems for major Staphylococcus aureus lineages. MBio 6:e00308-00315. https://doi.org/10.1128/mBio.00308-15

    Article  CAS  Google Scholar 

  26. Schenk S, Laddaga RA (1992) Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett 73:133–138

    Article  CAS  Google Scholar 

  27. Mosberg JA, Gregg CJ, Lajoie MJ et al (2012) Improving lambda red genome engineering in Escherichia coli via rational removal of endogenous nucleases. PLoS One 7:e44638. https://doi.org/10.1371/journal.pone.0044638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wang HH, Isaacs FJ, Carr PA et al (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894–898. https://doi.org/10.1038/nature08187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sawitzke JA, Costantino N, Li X-T et al (2011) Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering. J Mol Biol 407:45–59. https://doi.org/10.1016/j.jmb.2011.01.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yampolsky LY, Stoltzfus A (2005) The exchangeability of amino acids in proteins. Genetics 170:1459–1472. https://doi.org/10.1534/genetics.104.039107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ausubel FM (1987) Current protocols in molecular biology. Wiley, Brooklyn, NY

    Google Scholar 

  32. Liu J, Huang S, Sun M et al (2012) An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods 8:34. https://doi.org/10.1186/1746-4811-8-34

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Moreno-Mateos MA, Vejnar CE, Beaudoin J-D et al (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12:982–988. https://doi.org/10.1038/nmeth.3543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA

    Kelsi Penewit & Stephen J. Salipante

Authors
  1. Kelsi Penewit
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  2. Stephen J. Salipante
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Corresponding author

Correspondence to Stephen J. Salipante .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA

    Christopher R. Reisch

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Penewit, K., Salipante, S.J. (2022). Recombineering in Staphylococcus aureus. In: Reisch, C.R. (eds) Recombineering. Methods in Molecular Biology, vol 2479. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2233-9_10

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  • DOI: https://doi.org/10.1007/978-1-0716-2233-9_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2232-2

  • Online ISBN: 978-1-0716-2233-9

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