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Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome

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Cite this article as: eLife 2019;8:e42549 doi: 10.7554/eLife.42549

Abstract

Current technologies to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology to efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents that uncouples sequence diversity from sequence distribution. We demonstrate fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed at generating the up-to-date largest gRNA library to simultaneously interrogate the coding and noncoding human genome and identify genes, predicted promoter flanking regions, transcription factor and CTCF binding sites linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

Data availability

All data generated or analysed during this study are included in the manuscript, supplementary files or are available through GitHub or Dryad. NGS data and custom software is available as supplementary files and from Dryad and GitHub. Plasmids encoding oTGW 3Cs-gRNA libraries will be made available through the Goethe University Depository (http://innovectis.de/technologien/goethe-depository/).

The following data sets were generated

Article and author information

Author details

  1. Martin Wegner

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    Martin Wegner, The Goethe University Frankfurt has filed a patent related to this work on which M.W. is an inventor (WO2017EP84625).
  2. Valentina Diehl

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    Valentina Diehl, The Goethe University Frankfurt has filed a patent related to this work on which V.D. is an inventor (WO2017EP84625).
  3. Verena Bittl

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  4. Rahel de Bruyn

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    Rahel de Bruyn, The Goethe University Frankfurt has filed a patent related to this work on which R.D.B. is an inventor (WO2017EP84625).
  5. Svenja Wiechmann

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    Svenja Wiechmann, The Goethe University Frankfurt has filed a patent related to this work on which S.W. is an inventor (WO2017EP84625).
  6. Yves Matthess

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4040-1258
  7. Marie Hebel

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  8. Michael GB Hayes

    Department of Medicine, University of California, San Diego, La Jolla, United States
    Competing interests
    No competing interests declared.
  9. Simone Schaubeck

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  10. Christopher Benner

    Department of Medicine, University of California, San Diego, La Jolla, United States
    Competing interests
    No competing interests declared.
  11. Sven Heinz

    Department of Medicine, University of California, San Diego, La Jolla, United States
    Competing interests
    No competing interests declared.
  12. Anja Bremm

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1386-0926
  13. Ivan Dikic

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    Ivan Dikic, is co-founder, shareholder and CEO of Vivlion GmbH in Gründung. Also a senior editor of eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8156-9511
  14. Andreas Ernst

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    Competing interests
    Andreas Ernst, The Goethe University Frankfurt has filed a patent related to this work on which A.E. is an inventor (WO2017EP84625).
  15. Manuel Kaulich

    Institute of Biochemistry II, Goethe University Frankfurt, Frankfurt am Main, Germany
    For correspondence
    kaulich@em.uni-frankfurt.de
    Competing interests
    Manuel Kaulich, The Goethe University Frankfurt has filed a patent related to this work on which M.K. is an inventor (WO2017EP84625). Also co-founder, shareholder and CSO of Vivlion GmbH in Gründung.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9528-8822

Funding

Hessisches Ministerium für Wissenschaft und Kunst (IIIL5-518/17.004)

  • Manuel Kaulich

Deutsche Forschungsgemeinschaft (EXC115/2)

  • Manuel Kaulich

Hessisches Ministerium für Wissenschaft und Kunst (IIIL5-519/03/03.001)

  • Manuel Kaulich

Deutsche Forschungsgemeinschaft (EXC147/2)

  • Manuel Kaulich

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jonathan S Weissman, University of California, San Francisco, United States

Publication history

  1. Received: October 7, 2018
  2. Accepted: February 25, 2019
  3. Accepted Manuscript published: March 6, 2019 (version 1)
  4. Version of Record published: March 19, 2019 (version 2)

Copyright

© 2019, Wegner et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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