In Vivo Site-Specific Mutagenesis and Gene Collage Using the Delitto Perfetto System in Yeast Saccharomyces cerevisiae

  • Samantha Stuckey
  • Kuntal Mukherjee
  • Francesca StoriciEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 745)


Delitto perfetto is a site-specific in vivo mutagenesis system that has been developed to generate changes at will in the genome of the yeast Saccharomyces cerevisiae. Using this technique, it is possible to rapidly and efficiently engineer yeast strains without requiring several intermediate steps as it functions in only two steps, both of which rely on homologous recombination to drive the changes to the target DNA region. The first step involves the insertion of a cassette containing two markers at or near the locus to be altered. The second step involves complete removal of this cassette with oligonucleotides and/or other genetic material and transfer of the expected genetic modification(s) to the chosen DNA locus. Here we provide a detailed protocol of the delitto perfetto approach and present examples of the most common and useful applications for in vivo mutagenesis to generate base substitutions, deletions, insertions, as well as for precise in vivo assembly and integration of multiple genetic elements, or gene collage.

Key words

DNA modification DNA oligonucleotides site-directed mutagenesis gene targeting delitto perfetto system double-strand break yeast Saccharomyces cerevisiae gene collage 



We thank the members of our lab for their contributions to the editing and revision of this work, notably Rekha Pai, Patrick Ruff, and Ying Shen. We also thank Lee Katz for assistance in proofreading and revision. This work was funded in part by the Georgia Cancer Coalition grant R9028 and the NIH R21EB9228.


  1. 1.
    Sherman, F. (2002) Getting started with yeast. Methods Enzymol 350, 3–41.PubMedCrossRefGoogle Scholar
  2. 2.
    Dujon, B. (1996) The yeast genome project: what did we learn? Trends Genet 12, 263–270.PubMedCrossRefGoogle Scholar
  3. 3.
    Oliver, S.G. (1996) From DNA sequence to biological function. Nature 379, 653–654.CrossRefGoogle Scholar
  4. 4.
    Winzeler, E.A., and Davis, R.W. (1997) Functional analysis of the yeast genome. Curr Opin Genet Dev 7, 771–776.PubMedCrossRefGoogle Scholar
  5. 5.
    Resnick, M.A., and Cox, B.S. (2000) Yeast as an honorary mammal. Mutat Res 451, 1–11.PubMedGoogle Scholar
  6. 6.
    Wach, A., Brachat, A., Pohlmann, R., and Philippsen, P. (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.PubMedCrossRefGoogle Scholar
  7. 7.
    Storici, F., Lewis, L.K., and Resnick, M.A. (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol 19, 773–776.PubMedCrossRefGoogle Scholar
  8. 8.
    Storici, F., and Resnick, M.A. (2003) Delitto perfetto targeted mutagenesis in yeast with oligonucleotides. In Genetic engineering, principle and methods, Vol. 25 J.K. Setlow, ed. (Upton, NY: Kluwer Academic/Plenum Publisher), pp. 189–207.Google Scholar
  9. 9.
    Storici, F., Durham, C., Gordenin, D., and Resnick, M. (2003) Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. Proc Natl Acad Sci 100, 14994–14999.PubMedCrossRefGoogle Scholar
  10. 10.
    Storici, F., Snipe, J., Chan, G., Gordenin, G., and Resnick, M. (2006) Conservative repair of a chromosomal double-strand break by single-strand DNA through two steps of annealing. Mol Cell Biol 26, 7645–7657.PubMedCrossRefGoogle Scholar
  11. 11.
    Storici, F., and Resnick, M. (2006) The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol 409, 329–345.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Samantha Stuckey
    • 1
  • Kuntal Mukherjee
    • 1
  • Francesca Storici
    • 1
    Email author
  1. 1.School of Biology, Georgia Institute of TechnologyAtlantaUSA

Personalised recommendations