Skip to main content
SpringerLink
Log in

Genetic Modification Stimulated by the Induction of a Site-Specific Break Distant from the Locus of Correction in Haploid and Diploid Yeast Saccharomyces cerevisiae

  • Protocol
  • First Online:
Gene Correction

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1114))

Abstract

Generation of a site-specific break at a genomic locus to stimulate homologous recombination (HR) is used in many organisms to efficiently target genes for various types of genetic modification. Additionally, a site-specific chromosomal break can be used to trigger HR at genomic regions distant from the break, thereby largely expanding the region available for introducing desired mutations. In contrast to the former approach, the latter presents an alternative way in which genes can be efficiently modified also when it is not possible or desirable to introduce a break in the vicinity of the targeting locus. This type of in vivo site-directed mutagenesis distant from a break can be accomplished in the yeast model organism Saccharomyces cerevisiae because the generation of a double-strand break (DSB) in yeast chromosomal DNA activates HR at long regions upstream and downstream from the break site. Here we provide a protocol for efficiently altering a yeast chromosomal locus following the induction of a DSB several kilobase pairs distant from the site of gene correction. The techniques described can be used in both diploid and haploid yeast strains, and we provide examples of the gene correction assays.

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

Access this chapter

Log in via an institution
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. Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301

    Article  CAS  PubMed  Google Scholar 

  2. Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512

    Article  CAS  PubMed  Google Scholar 

  3. Doetschman T, Maeda N, Smithies O (1988) Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci U S A 85:8583–8587

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Kuehn MR, Bradley A, Robertson EJ, Evans MJ (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326:295–298

    Article  CAS  PubMed  Google Scholar 

  5. Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–1206

    Article  CAS  PubMed  Google Scholar 

  6. Rong YS, Golic KG (2000) Gene targeting by homologous recombination in Drosophila. Science 288:2013–2018

    Article  CAS  PubMed  Google Scholar 

  7. Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300:763

    Article  PubMed  Google Scholar 

  8. Liang F, Han M, Romanienko PJ, Jasin M (1998) Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci U S A 95:5172–5177

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764

    Article  CAS  PubMed  Google Scholar 

  10. Vasquez KM, Marburger K, Intody Z, Wilson JH (2001) Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci U S A 98:8403–8410

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P, Paques F (2011) Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther 11:11–27

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Kumar S, Allen GC, Thompson WF (2006) Gene targeting in plants: fingers on the move. Trends Plant Sci 11:159–161

    Article  CAS  PubMed  Google Scholar 

  13. Donoho G, Jasin M, Berg P (1998) Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. Mol Cell Biol 18:4070–4078

    CAS  PubMed Central  PubMed  Google Scholar 

  14. Richardson C, Moynahan ME, Jasin M (1998) Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev 12:3831–3842

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Takata M, Sasaki MS, Sonoda E, Morrison C, Hashimoto M, Utsumi H, Yamaguchi-Iwai Y, Shinohara A, Takeda S (1998) Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 17:5497–5508

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Schaefer DG (2001) Gene targeting in Physcomitrella patens. Curr Opin Plant Biol 4:143–150

    Article  CAS  PubMed  Google Scholar 

  17. Storici F, Snipe JR, Chan GK, Gordenin DA, Resnick MA (2006) Conservative repair of a chromosomal double-strand break by single-strand DNA through two steps of annealing. Mol Cell Biol 26:7645–7657

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Yang Y, Sterling J, Storici F, Resnick MA, Gordenin DA (2008) Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae. PLoS Genet 4:e1000264

    Article  PubMed Central  PubMed  Google Scholar 

  19. Lee SE, Pellicioli A, Demeter J, Vaze MP, Gasch AP, Malkova A, Brown PO, Botstein D, Stearns T, Foiani M, Haber JE (2000) Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol 65:303–314

    Article  CAS  PubMed  Google Scholar 

  20. Storici F, Resnick MA (2006) The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol 409:329–345

    Article  CAS  PubMed  Google Scholar 

  21. Rahn JJ, Rowley B, Lowery MP, Coletta LD, Limanni T, Nairn RS, Adair GM (2011) Effects of varying gene targeting parameters on processing of recombination intermediates by ERCC1-XPF. DNA Repair (Amst) 10:188–198

    Article  CAS  Google Scholar 

  22. Storici F, Durham CL, Gordenin DA, Resnick MA (2003) Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. Proc Natl Acad Sci U S A 100:14994–14999

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651

    Article  CAS  PubMed  Google Scholar 

  24. Ira G, Pellicioli A, Balijja A, Wang X, Fiorani S, Carotenuto W, Liberi G, Bressan D, Wan L, Hollingsworth NM, Haber JE, Foiani M (2004) DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431:1011–1017

    Article  CAS  PubMed  Google Scholar 

  25. Perez C, Guyot V, Cabaniols JP, Gouble A, Micheaux B, Smith J, Leduc S, Paques F, Duchateau P (2005) Factors affecting double-strand break-induced homologous recombination in mammalian cells. Biotechniques 39:109–115

    Article  CAS  PubMed  Google Scholar 

  26. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785

    Article  CAS  PubMed  Google Scholar 

  27. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, Unger-Wallace E, Sander JD, Muller-Lerch F, Fu F, Pearlberg J, Gobel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, McCray PB Jr, Cathomen T, Voytas DF, Joung JK (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31:294–301

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29:731–734

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Stuckey S, Mukherjee K, Storici F (2011) In vivo site-specific mutagenesis and gene collage using the delitto perfetto system in yeast Saccharomyces cerevisiae. Methods Mol Biol 745:173–191

    Article  CAS  PubMed  Google Scholar 

  33. Storici F, Lewis LK, Resnick MA (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol 19:773–776

    Article  CAS  PubMed  Google Scholar 

  34. Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank all the members of the Storici lab for their suggestions and contributions to the editing and revision of this work. This work was supported by the Graduate Assistance in Areas of National Need (GAANN) fellowship to S.S., the NSF grant MCB-1021763 and the Georgia Cancer Coalition grant R9028 to F.S.

Author information

Authors and Affiliations

  1. School of Biology, Georgia Institute of Technology, Atlanta, GA, USA

    Samantha Stuckey & Francesca Storici

Authors
  1. Samantha Stuckey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca Storici
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Georgia Institute of Technology, Atlanta, Georgia, USA

    Francesca Storici

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Stuckey, S., Storici, F. (2014). Genetic Modification Stimulated by the Induction of a Site-Specific Break Distant from the Locus of Correction in Haploid and Diploid Yeast Saccharomyces cerevisiae . In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-761-7_20

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-760-0

  • Online ISBN: 978-1-62703-761-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation