Abstract

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

Data availability

The data that support the findings of this study are available within the paper and its Supplementary files. Source data files have been provided for Figure 4, Figure 5, Figure 6, Figure Supplement 1, Figure Supplement 2, and Figure Supplement 3.Scripts for mapping sequencing data, counting mutations and generating plots are available at https://github.com/HLindsay/Savic_CRISPR_HDR. Fastq files have been uploaded to ArrayExpress, and accession number is E-MTAB-6808.

The following data sets were generated

Article and author information

Author details

  1. Natasa Savic

    The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  2. Femke CAS Ringnalda

    The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Helen Lindsay

    The Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  4. Christian Berk

    Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Katja Bargsten

    Department of Biochemistry, University of Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Yizhou Li

    Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Dario Neri

    Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Mark D Robinson

    The Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  9. Constance Ciaudo

    The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0857-4506
  10. Jonathan Hall

    Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4160-7135
  11. Martin Jinek

    Department of Biochemistry, University of Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7601-210X
  12. Gerald Schwank

    The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland
    For correspondence
    schwankg@ethz.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0767-2953

Funding

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (PMPDP3_171388)

  • Natasa Savic

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_160230)

  • Gerald Schwank

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_149393)

  • Martin Jinek

Vallee Foundation

  • Martin Jinek

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

Reviewing Editor

  1. Bernard de Massy, Institute of Human Genetics, CNRS UPR 1142, France

Version history

  1. Received: November 23, 2017
  2. Accepted: May 26, 2018
  3. Accepted Manuscript published: May 29, 2018 (version 1)
  4. Version of Record published: June 28, 2018 (version 2)
  5. Version of Record updated: June 29, 2018 (version 3)
  6. Version of Record updated: July 4, 2018 (version 4)
  7. Version of Record updated: March 6, 2019 (version 5)

Copyright

© 2018, Savic 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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  1. Natasa Savic
  2. Femke CAS Ringnalda
  3. Helen Lindsay
  4. Christian Berk
  5. Katja Bargsten
  6. Yizhou Li
  7. Dario Neri
  8. Mark D Robinson
  9. Constance Ciaudo
  10. Jonathan Hall
  11. Martin Jinek
  12. Gerald Schwank
(2018)
Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
eLife 7:e33761.
https://doi.org/10.7554/eLife.33761

Share this article

https://doi.org/10.7554/eLife.33761

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