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Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering

Nature Biotechnology volume 35, pages 4855 (2017) | Download Citation

Abstract

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR–Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype–phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

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Acknowledgements

This work would not have been possible without the insights and efforts of a number of talented individuals. We would like to thank T. Mansell, N. Boyle, K. Fujimori, and H. Chilton for their input, feedback, guidance and contributions to this work. This work was supported by the US Department of Energy (Grant DE-SC0008812) and CAPES foundation (grant #0315133).

Author information

Affiliations

  1. Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.

    • Andrew D Garst
    • , Marcelo C Bassalo
    • , Gur Pines
    • , Sean A Lynch
    • , Andrea L Halweg-Edwards
    • , Rongming Liu
    • , Liya Liang
    • , Zhiwen Wang
    • , Ramsey Zeitoun
    •  & Ryan T Gill
  2. Muse Biotechnology Inc., Boulder, Colorado, USA.

    • Andrew D Garst
    • , Andrea L Halweg-Edwards
    •  & William G Alexander
  3. Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, USA.

    • Marcelo C Bassalo
  4. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, People's Republic of China.

    • Zhiwen Wang
  5. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, People's Republic of China.

    • Zhiwen Wang
  6. Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, People's Republic of China.

    • Zhiwen Wang

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Contributions

R.T.G., M.C.B., G.P., S.A.L. and R.Z. all contributed to A.D.G.'s development of the concept. A.D.G., M.C.B., G.P., S.A.L. and R.T.G. all aided in the design of experiments. Scripts to automate CREATE cassette design were written by A.D.G. Library construction and recombineering was done by A.D.G. Selections, sample preparation, sequencing, clonal reconstructions and growth validations of selected variants were done by A.D.G., M.C.B., G.P., R.L. and Z.W. Sequencing data analysis was done by A.D.G. with contributions to the statistical analysis provided by R.Z. and R.T.G. The http://www.thebioverse.org web interface was developed by A.L.H.-E. Yeast validation of CREATE methodology was performed by L.L., R.L. and W.G.A. The manuscript was written by A.D.G. and R.T.G.

Competing interests

R.T.G. and A.D.G. have a patent application pending (WO/2015/123339) whose value may be affected by the publication of this paper. R.T.G., A.D.G. and A.L.H.-E. have financial interests in Muse Biotechnology Inc., which is commercializing the CREATE technology.

Corresponding author

Correspondence to Ryan T Gill.

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https://doi.org/10.1038/nbt.3718

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