Skip to main content

Mutagenic Inverted Repeats Assisted Genome Engineering (MIRAGE) in Saccharomyces cerevisiae: Deletion of gal7

  • Protocol
  • First Online:
Microbial Metabolic Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 834))

Abstract

MIRAGE is a unique in vivo genome editing technique that exploits the inherent instability of inverted repeats (palindromes) in the Saccharomyces cerevisiae chromosome. As a technique able to quickly create deletions as well as precise point mutations, it is valuable in applications that require creation of designer strains of this yeast. In particular, it has various potential applications in metabolic engineering, systems biology, synthetic biology, and molecular genetics.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gibson, D.G., Benders, G.A., Andrews-Pfannkoch, C., Denisova, E.A., Baden-Tillson, H., Zaveri, J., Stockwell, T.B., Brownley, A., Thomas, D.W., Algire, M.A. et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science, 319, 1215–1220.

    Article  PubMed  CAS  Google Scholar 

  2. Lartigue, C., Vashee, S., Algire, M.A., Chuang, R.Y., Benders, G.A., Ma, L., Noskov, V.N., Denisova, E.A., Gibson, D.G., Assad-Garcia, N. et al. (2009) Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science, 325, 1693–1696.

    Article  PubMed  CAS  Google Scholar 

  3. Scherer, S. and Davis, R.W. (1979) Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci USA, 76, 4951–4955.

    Article  PubMed  CAS  Google Scholar 

  4. Gray, M., Piccirillo, S. and Honigberg, S.M. (2005) Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome. FEMS Microbiol Lett, 248, 31–36.

    Article  PubMed  CAS  Google Scholar 

  5. Storici, F., Durham, C.L., Gordenin, D.A. and Resnick, M.A. (2003) Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. Proc Natl Acad Sci USA, 100, 14994–14999.

    Article  PubMed  CAS  Google Scholar 

  6. Storici, F., Lewis, L.K. and Resnick, M.A. (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat. Biotechnol, 19, 773–776.

    Article  PubMed  CAS  Google Scholar 

  7. Langle-Rouault, F. and Jacobs, E. (1995) A method for performing precise alterations in the yeast genome using a recycable selectable marker. Nucleic Acids Res. 23, 3079–3081.

    Article  PubMed  CAS  Google Scholar 

  8. Nair, N.U. and Zhao, H. (2009) Mutagenic inverted repeat assisted genome engineering (MIRAGE). Nucleic Acids Res. 37, e9.

    Article  PubMed  Google Scholar 

  9. Gordenin, D.A., Lobachev, K.S., Degtyareva, N.P., Malkova, A.L., Perkins, E. and Resnick, M.A. (1993) Inverted DNA repeats: a source of eukaryotic genomic instability. Mol. Cell Biol. 13, 5315–5322.

    PubMed  CAS  Google Scholar 

  10. Lobachev, K.S., Shor, B.M., Tran, H.T., Taylor, W., Keen, J.D., Resnick, M.A. and Gordenin, D.A. (1998) Factors affecting inverted repeat stimulation of recombination and deletion in Saccharomyces cerevisiae. Genetics, 148, 1507–1524.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Huimin Zhao .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Nair, N.U., Zhao, H. (2012). Mutagenic Inverted Repeats Assisted Genome Engineering (MIRAGE) in Saccharomyces cerevisiae: Deletion of gal7 . In: Cheng, Q. (eds) Microbial Metabolic Engineering. Methods in Molecular Biology, vol 834. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-483-4_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-483-4_5

  • Published:

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-482-7

  • Online ISBN: 978-1-61779-483-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics