Letter | Published:

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast

Nature Biotechnology volume 36, pages 540546 (2018) | Download Citation

Abstract

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80–100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

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Acknowledgements

G.M.C. was supported by NIH grants RM1 HG008525 and P50 HG005550. A.C. was funded by the National Cancer Institute grant no. 5T32CA009216-34. J.J.C. was funded by the Defense Threat Reduction Agency grant HDTRA1-14-1-0006, the Paul G. Allen Frontiers Group. Y.Y. was supported by the Damon Runyon Research Foundation grant DRG-2248-16.

Author information

Author notes

    • Alejandro Chavez

    Present address: Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA.

    • Xiaoge Guo
    • , Alejandro Chavez
    •  & Angela Tung

    These authors contributed equally to this work.

Affiliations

  1. Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA.

    • Xiaoge Guo
    • , Alejandro Chavez
    • , Angela Tung
    • , Yingleong Chan
    • , Christian Kaas
    • , Ryan Cecchi
    • , Santiago Lopez Garnier
    • , Eric D Kelsic
    • , Max Schubert
    • , James E DiCarlo
    • , James J Collins
    •  & George M Church
  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Xiaoge Guo
    • , Alejandro Chavez
    • , Yingleong Chan
    • , Christian Kaas
    • , Eric D Kelsic
    • , Max Schubert
    • , James E DiCarlo
    •  & George M Church
  3. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Alejandro Chavez
  4. Department of Expression Technologies 2, Novo Nordisk A/S, Maaloev, Denmark.

    • Christian Kaas
  5. Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

    • Yi Yin
  6. Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, USA.

    • James E DiCarlo
  7. Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • James J Collins
  8. Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • James J Collins
  9. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • James J Collins
  10. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • James J Collins

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Contributions

X.G. and A.C. conceived the idea, led the study, and designed all experiments. A.C. and R.C. with input from J.E.D. demonstrated the initial feasibility of the guide+donor approach. X.G. performed majority of the experiments, including the oligo library design, library construction and analysis, with significant technical contribution from A.T. Y.C. provided expertise in statistical analysis. Y.Y. performed the whole genome sequencing experiment for off-target analysis. C.K. generated the RNA-seq data for the BY4741 yeast strain, provided the FPKM values and analyzed the whole genome data from yeast isolates modified by guide+donor for off-target effects. S.L.G. assisted with oligo library design. E.K. provided insight with regard to library construction methods and analysis. M.S. provided technical expertise with regard to methods to increase guide+donor efficiency. J.J.C. and G.M.C. oversaw the study. X.G. and A.C. wrote the manuscript with input from all authors.

Competing interests

G.M.C. is the founder and holds leadership positions in many companies (http://arep.med.harvard.edu/gmc/tech.html). X.G., A.C., M.S., and E.K. have filed a patent application (US Patent Application 62/348,438) relating to this work.

Corresponding authors

Correspondence to Alejandro Chavez or George M Church.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Tables

    Supplementary tables1–7

Excel files

  1. 1.

    Supplementary Data 1

    Log2-fold changes in abundance of guide+donor members in SGS1 tiling deletion screen

  2. 2.

    Supplementary Data 2

    Log2-fold changes in abundance of guide+donor members in Sgs1 conserved residue amino acid substitution screen

  3. 3.

    Supplementary Data 3

    Log2-fold changes in abundance of guide+donor members in smORF screen

  4. 4.

    Supplementary Data 4

    Analysis of amino acid length, gene expression, prediction of secondary structure formation and level of conservation in humans between smORFs and ORFs

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DOI

https://doi.org/10.1038/nbt.4147

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