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

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

Abstract

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 

Notes

Acknowledgments

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).

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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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