Brief Communication

Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision

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Abstract

We developed a CRISPR–Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.

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Sequence Read Archive

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Acknowledgements

This work was supported by the Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign and the US Department of Energy (DE-SC0018260). We thank A. Hernandez and C. Wright for assistance with next-generation sequencing, J. Zadeh for assistance with NGS data processing and analysis.

Author information

Author notes

    • Han Xiao

    Present address: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and Laboratory of Molecular Biochemical Engineering, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

Affiliations

  1. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Zehua Bao
    • , Han Xiao
    •  & Huimin Zhao
  2. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Mohammad HamediRad
    • , Pu Xue
    • , Ran Chao
    •  & Huimin Zhao
  3. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Ipek Tasan
    •  & Huimin Zhao
  4. Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore.

    • Jing Liang
    •  & Huimin Zhao
  5. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Huimin Zhao
  6. Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Huimin Zhao

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Contributions

Z.B. and H.Z. conceived this project. Z.B., M.H., and H.X. designed the CHAnGE cassettes. R.C. and J.L. generated the ORF list and all possible guide sequences. M.H. sorted and selected the guide and homology arm sequences. Z.B., P.X., and I.T. performed the experiments. Z.B. analyzed the data. H.Z. supervised the research. Z.B. and H.Z. wrote the manuscript.

Competing interests

A patent application has been filed on this technology, on which H.Z. and Z.B. are authors.

Corresponding author

Correspondence to Huimin Zhao.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Notes 1 and 2, Supplementary Figures 1–18, and Supplementary Tables 1, 2, 4, 5, 7.

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Code

    Supplementary Code

Excel files

  1. 1.

    Supplementary Table 3

    A summary of 24865 CHAnGE cassette sequences.

  2. 2.

    Supplementary Table 6

    A summary of 580 SIZ1 CHAnGE cassette sequences.