Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae

  • Shuobo Shi
  • Youyun Liang
  • Ee Lui Ang
  • Huimin ZhaoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1927)


Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) to enable unprecedented high-efficiency, multicopy, markerless integrations of large biochemical pathways into the S. cerevisiae genome. Detailed protocols are provided on the entire workflow which includes pDi-CRISPR plasmid and donor DNA construction, Di-CRISPR-mediated integration and analysis of integration efficiencies and copy numbers through flow cytometry and quantitative polymerase chain reaction (qPCR).

Key words

Delta integration CRISPR-Cas Saccharomyces cerevisiae Genome integration Genome engineering Synthetic biology 



We thank Agency for Science, Technology, and Research, Singapore for supporting various research projects in the Metabolic Engineering Research Laboratory (MERL) through the Visiting Investigator Programme to H.Z (1535j00137).


  1. 1.
    Hong KK, Nielsen J (2012) Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol Life Sci 69:2671–2690CrossRefGoogle Scholar
  2. 2.
    Nevoigt E (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 72(3):379–412CrossRefGoogle Scholar
  3. 3.
    Karim AS, Curran KA, Alper HS (2013) Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res 13(1):107–116CrossRefGoogle Scholar
  4. 4.
    Da Silva NA, Srikrishnan S (2012) Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae. FEMS Yeast Res 12(2):197–214CrossRefGoogle Scholar
  5. 5.
    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(25):14994–14999CrossRefGoogle Scholar
  6. 6.
    de Jong B, Shi S, Valle-Rodríguez J, Siewers V, Nielsen J (2015) Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration. J Ind Microbiol Biotechnol 42(3):477–486CrossRefGoogle Scholar
  7. 7.
    Lee FWF, Silva NAD (1997) Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 48(3):339–345CrossRefGoogle Scholar
  8. 8.
    Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2010) Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb Cell Factories 9:32CrossRefGoogle Scholar
  9. 9.
    Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186(2):757–761CrossRefGoogle Scholar
  10. 10.
    Li T, Huang S, Zhao X, Wright DA, Carpenter S, Spalding MH et al (2011) Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 39(14):6315–6325CrossRefGoogle Scholar
  11. 11.
    Jakočiūnas T, Bonde I, Herrgård M, Harrison SJ, Kristensen M, Pedersen LE et al (2015) Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae. Metab Eng 28:213–222CrossRefGoogle Scholar
  12. 12.
    DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343CrossRefGoogle Scholar
  13. 13.
    Bao Z, Xiao H, Liang J, Zhang L, Xiong X, Sun N et al (2015) Homology-integrated CRISPR–Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth Biol 4(5):585–594CrossRefGoogle Scholar
  14. 14.
    Shi S, Liang Y, Zhang MM, Ang EL, Zhao H (2016) A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae. Metab Eng 33:19–27CrossRefGoogle Scholar
  15. 15.
    Luo Y, Huang H, Liang J, Wang M, Lu L, Shao Z et al (2013) Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster. Nat Commun 4:2894CrossRefGoogle Scholar
  16. 16.
    Sun J, Shao Z, Zhao H, Nair N, Wen F, Xu J-H et al (2012) Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng 109(8):2082–2092CrossRefGoogle Scholar
  17. 17.
    Du J, Yuan Y, Si T, Lian J, Zhao H (2012) Customized optimization of metabolic pathways by combinatorial transcriptional engineering. Nucleic Acids Res 40(18):e142–e142CrossRefGoogle Scholar
  18. 18.
    Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34CrossRefGoogle Scholar
  19. 19.
    Whelan JA, Russell NB, Whelan MA (2003) A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 278(1):261–269CrossRefGoogle Scholar
  20. 20.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics methods and protocols: methods in molecular biology. Edited by Krawetz S, Misener S. Humana Press, Totowa, NJ, pp 365–386Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Shuobo Shi
    • 1
  • Youyun Liang
    • 1
  • Ee Lui Ang
    • 1
  • Huimin Zhao
    • 1
    • 2
    Email author
  1. 1.Metabolic Engineering Research Laboratory, Science and Engineering InstitutesAgency for Science, Technology and ResearchSingaporeSingapore
  2. 2.Department of Chemical and Biomolecular EngineeringUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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