Abstract

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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References

  1. 1.

    et al. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nat. Biotechnol. 35, 48–55 (2017).

  2. 2.

    et al. Highly parallel genome variant engineering with CRISPR/Cas9 in eukaryotic cells. Preprint at (2017).

  3. 3.

    et al. High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9. Preprint at (2017).

  4. 4.

    et al. CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens. Nat. Methods 14, 1191–1197 (2017).

  5. 5.

    et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

  6. 6.

    & The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Curr. Genet. 30, 485–492 (1996).

  7. 7.

    et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).

  8. 8.

    et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

  9. 9.

    , , & Homologous recombinational repair of double-strand breaks in yeast is enhanced by MAT heterozygosity through yKU-dependent and -independent mechanisms. Genetics 157, 579–589 (2001).

  10. 10.

    & A 700 bp cis-acting region controls mating-type dependent recombination along the entire left arm of yeast chromosome III. Cell 87, 277–285 (1996).

  11. 11.

    , , , & Saccharomyces forkhead protein Fkh1 regulates donor preference during mating-type switching through the recombination enhancer. Genes Dev. 16, 2085–2096 (2002).

  12. 12.

    et al. Binding of the Fkh1 Forkhead Associated Domain to a phosphopeptide within the Mph1 DNA helicase regulates mating-type switching in budding yeast. PLOS Genet. 12, e1006094 (2016).

  13. 13.

    et al. Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1. PLoS Genet. 8, e1002630 (2012).

  14. 14.

    et al. SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans. Mol. Microbiol. 80, 1088–1101 (2011).

  15. 15.

    et al. PITPs as targets for selectively interfering with phosphoinositide signaling in cells. Nat. Chem. Biol. 10, 76–84 (2014).

  16. 16.

    et al. Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis. EMBO J. 15, 6447–6459 (1996).

  17. 17.

    et al. Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex. J. Cell Biol. 157, 63–77 (2002).

  18. 18.

    et al. Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell 64, 789–800 (1991).

  19. 19.

    , , & Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo. Nucleic Acids Res. 44, 5365–5377 (2016).

  20. 20.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

  21. 21.

    , & Transcription termination by the eukaryotic RNA polymerase III. Biochim. Biophys. Acta 1829, 318–330 (2013).

  22. 22.

    , & Sequence context effects on oligo(dT) termination signal recognition by Saccharomyces cerevisiae RNA polymerase III. J. Biol. Chem. 280, 19551–19562 (2005).

  23. 23.

    et al. Widespread occurrence of non-canonical transcription termination by human RNA polymerase III. Nucleic Acids Res. 39, 5499–5512 (2011).

  24. 24.

    et al. A method for high-throughput production of sequence-verified DNA libraries and strain collections. Mol. Syst. Biol. 13, 913 (2017).

  25. 25.

    et al. Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair. Preprint at (2017).

  26. 26.

    et al. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res. 27, 578–581 (2017).

  27. 27.

    , & Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR. Preprint at (2017).

  28. 28.

    & Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev. 25, 409–433 (2011).

  29. 29.

    et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407–410 (2017).

  30. 30.

    et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

  31. 31.

    et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

  32. 32.

    et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

  33. 33.

    et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3, e03703 (2014).

  34. 34.

    , , , & Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

  35. 35.

    & High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

  36. 36.

    & SICtools: Find SNV/Indel differences between two bam files with near relationship. R package version 1.8.0. (2014). .

  37. 37.

    , , & Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol-transfer protein. Nature 391, 506–510 (1998).

  38. 38.

    , , , & Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748 (1997).

  39. 39.

    R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2018).

  40. 40.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  41. 41.

    , & Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012).

  42. 42.

    ggplot2: Elegant Graphics for Data Analysis (Springer, 2009).

  43. 43.

    Matplotlib: A 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

  44. 44.

    et al. seaborn: v0.7.1 (June 2016) (Zenodo, 2016). doi:10.5281/zenodo.54844.

Download references

Acknowledgements

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.

Affiliations

  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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Contributions

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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DOI

https://doi.org/10.1038/nbt.4137