Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR–Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide–donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA–Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

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This work was supported by grants from the US National Institutes of Health (P01HG000205 to L.M.S. and R.W.D., R01GM121932-01A1 to R.P.S., U01GM110706-02 to R.W.D., RO1GM61766 to J.E.H., and RO1GM44530 to V.A.B.), the National Institute of Standards and Technology (70NANB15H268 to M.L.S.), and the European Research Council Advanced Investigator Grant (AdG-294542 to L.M.S.). K.R.R. was supported by a National Research Council postdoctoral fellowship. A.T. and V.A.B. were supported by the Robert A. Welch Foundation (award BE-0017). S.C.V. was supported by a Swiss National Science Foundation postdoctoral fellowship (P2EZP3_165220). Certain commercial equipment, instruments, or materials are identified in this document. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the products identified are necessarily the best available for the purpose. We thank the EMBL Genomics Core Facility for support and optimization of barcode sequencing protocols. This work is dedicated to the memory of Joe Horecka (12/1/1963-10/20/2017).

Author information

Author notes

    • Kevin R Roy
    • , Justin D Smith
    •  & Sibylle C Vonesch

    These authors contributed equally to this work.


  1. Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA.

    • Kevin R Roy
    • , Justin D Smith
    • , Angela Chu
    • , Sundari Suresh
    • , Michelle Nguyen
    • , Joe Horecka
    • , Wallace T Burnett
    • , Maddison A Morgan
    • , Julia Schulz
    • , Kevin M Orsley
    • , Wu Wei
    • , Raeka S Aiyar
    • , Ronald W Davis
    • , Robert P St.Onge
    •  & Lars M Steinmetz
  2. Genome-Scale Measurements Group, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.

    • Kevin R Roy
    •  & Marc L Salit
  3. Joint Initiative for Metrology in Biology, Stanford, California, USA.

    • Kevin R Roy
    • , Marc L Salit
    •  & Lars M Steinmetz
  4. Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.

    • Kevin R Roy
    • , Justin D Smith
    • , Michelle Nguyen
    • , Wallace T Burnett
    • , Maddison A Morgan
    • , Julia Schulz
    • , Kevin M Orsley
    • , Wu Wei
    • , Ronald W Davis
    •  & Lars M Steinmetz
  5. European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.

    • Sibylle C Vonesch
    • , Gen Lin
    • , Chelsea Szu Tu
    • , Alex R Lederer
    •  & Lars M Steinmetz
  6. Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.

    • Angela Chu
    • , Sundari Suresh
    • , Joe Horecka
    • , Ronald W Davis
    •  & Robert P St.Onge
  7. Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA.

    • Ashutosh Tripathi
    •  & Vytas A Bankaitis
  8. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.

    • Vytas A Bankaitis
  9. Department of Chemistry, Texas A&M University, College Station, Texas, USA.

    • Vytas A Bankaitis
  10. Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts, USA.

    • James E Haber


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K.R.R., J.D.S., S.C.V., R.P.S., and L.M.S. conceived and designed the study, and wrote and edited the paper. K.R.R., J.D.S., S.C.V., and R.P.S. performed experiments and analyzed data. K.R.R., S.C.V., G.L., and A.R.L. analyzed NGS data; C.S.T., A.C., S.S., M.N., J.H., W.T.B., M.A.M., J.S., and K.M.O. performed experiments. A.T. and V.A.B. performed computational structural analysis on Sec14p-NPPM; W.W. performed variant calling for the different yeast strains. J.E.H. suggested adapting the LexA–Fkh1p system to the guide–donor plasmid. R.S.A., R.W.D., and M.L.S. advised the study. R.P.S. and L.M.S. were responsible for the coordination of the study. All authors read, corrected, and approved the final manuscript.

Competing interests

K.R.R., J.D.S., J.E.H., R.P.S. and L.M.S. have filed a provisional application (US 62/559,493) with the US Patent and Trademark Office on this work.

Corresponding authors

Correspondence to Robert P St.Onge or Lars M Steinmetz.

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