Rapid genome editing by CRISPR-Cas9-POLD3 fusion

  1. Ganna Reint
  2. Zhuokun Li
  3. Kornel Labun
  4. Salla Keskitalo
  5. Inkeri Soppa
  6. Katariina Mamia
  7. Eero Tolo
  8. Monika Szymanska
  9. Leonardo A Meza-Zepeda
  10. Susanne Lorenz
  11. Artur Cieslar-Pobuda
  12. Xian Hu
  13. Diana L Bordin
  14. Judith Staerk
  15. Eivind Valen
  16. Bernhard Schmierer
  17. Markku Varjosalo
  18. Jussi Taipale
  19. Emma Haapaniemi  Is a corresponding author
  1. University of Oslo, Norway
  2. University of Bergen, Norway
  3. University of Helsinki, Finland
  4. University of Oslo, Finland
  5. Oslo University Hospital, Norway
  6. Akershus University Hospital, Norway
  7. Karolinska Institute, Sweden
  8. University of Cambridge, United Kingdom

Abstract

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

Data availability

The following data sets were generated Reint et. al., (2021), Sequence Read Archive (SRA), BioProject ID: PRJNA782085. The following previously published data sets were used: Tsai et al., (2015) Sequence Read Archive (SRA), SRP050338. Custom scripts used to extract UMI from the demultiplexed fastq reads for the GUIDE-Seq analysis is publicly available at: https://bitbucket.org/valenlab/guide-seq-pold3. Sequences of Cas9 nuclease and GFP-BFP reporter cassette used in this study are available in Supplementary file 8.

Article and author information

Author details

  1. Ganna Reint

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4823-5485
  2. Zhuokun Li

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7297-6916
  3. Kornel Labun

    Department of Informatics, Computational Biology Unit, University of Bergen, Bergen, Norway
    Competing interests
    The authors declare that no competing interests exist.
  4. Salla Keskitalo

    Centre for Biotechnology, University of Helsinki, Helsinki, Finland
    Competing interests
    The authors declare that no competing interests exist.
  5. Inkeri Soppa

    Centre for Molecular Medicine, University of Oslo, Oslo, Finland
    Competing interests
    The authors declare that no competing interests exist.
  6. Katariina Mamia

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
  7. Eero Tolo

    Faculty of Social Sciences, University of Helsinki, Oslo, Finland
    Competing interests
    The authors declare that no competing interests exist.
  8. Monika Szymanska

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0957-9568
  9. Leonardo A Meza-Zepeda

    Department of Core Facilities, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
  10. Susanne Lorenz

    Department of Core Facilities, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
  11. Artur Cieslar-Pobuda

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
  12. Xian Hu

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3381-7514
  13. Diana L Bordin

    Department of Clinical Molecular Biology, Akershus University Hospital, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
  14. Judith Staerk

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8698-6998
  15. Eivind Valen

    Center for Biotechnology, University of Bergen, Bergen, Norway
    Competing interests
    The authors declare that no competing interests exist.
  16. Bernhard Schmierer

    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9082-7022
  17. Markku Varjosalo

    Centre for Biotechnology, University of Helsinki, Helsinki, Finland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1340-9732
  18. Jussi Taipale

    Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4204-0951
  19. Emma Haapaniemi

    Centre for Molecular Medicine, University of Oslo, Oslo, Norway
    For correspondence
    emma.haapaniemi@ncmm.uio.no
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6693-8208

Funding

Barncancerfonden

  • Kornel Labun

Norwegian Research Council

  • Emma Haapaniemi

South-Eastern Norway Regional Health Authority ((Grant no. 279922 to Hilde Nilsen))

  • Emma Haapaniemi

Knut och Alice Wallenbergs Stiftelse

  • Jussi Taipale

Cancerfonden

  • Emma Haapaniemi

Barncancerfonden

  • Emma Haapaniemi

Instrumentariumin Tiedesäätiö

  • Emma Haapaniemi

Science for Life Laboratory

  • Bernhard Schmierer

Academy of Finland

  • Markku Varjosalo
  • Jussi Taipale
  • Emma Haapaniemi

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

Reviewing Editor

  1. Jessica K Tyler, Weill Cornell Medicine, United States

Version history

  1. Received: November 13, 2021
  2. Accepted: November 15, 2021
  3. Accepted Manuscript published: December 13, 2021 (version 1)
  4. Version of Record published: January 10, 2022 (version 2)
  5. Version of Record updated: May 27, 2022 (version 3)

Copyright

© 2021, Reint 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. Ganna Reint
  2. Zhuokun Li
  3. Kornel Labun
  4. Salla Keskitalo
  5. Inkeri Soppa
  6. Katariina Mamia
  7. Eero Tolo
  8. Monika Szymanska
  9. Leonardo A Meza-Zepeda
  10. Susanne Lorenz
  11. Artur Cieslar-Pobuda
  12. Xian Hu
  13. Diana L Bordin
  14. Judith Staerk
  15. Eivind Valen
  16. Bernhard Schmierer
  17. Markku Varjosalo
  18. Jussi Taipale
  19. Emma Haapaniemi
(2021)
Rapid genome editing by CRISPR-Cas9-POLD3 fusion
eLife 10:e75415.
https://doi.org/10.7554/eLife.75415

Share this article

https://doi.org/10.7554/eLife.75415

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