Abstract

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straight-forward purification. Immediate activity is usually granted by administration of pre-assembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Thomas Thumberger

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    Thomas Thumberger, patent application pending (EP21166099.8) related to the findings described.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8485-457X
  2. Tinatini Tavhelidse-Suck

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    Tinatini Tavhelidse-Suck, patent application pending (EP21166099.8) related to the findings described.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6103-9019
  3. Jose Arturo Gutierrez-Triana

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    Jose Arturo Gutierrez-Triana, patent application pending (EP21166099.8) related to the findings described.
  4. Alex Cornean

    Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3727-7057
  5. Rebekka Medert

    Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  6. Bettina Welz

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  7. Marc Freichel

    Institute of Pharmacology, Heidelberg University, Heidelberg, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1387-2636
  8. Joachim Wittbrodt

    Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany
    For correspondence
    jochen.wittbrodt@cos.uni-heidelberg.de
    Competing interests
    Joachim Wittbrodt, patent application pending (EP21166099.8) related to the findings described.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8550-7377

Funding

Deutsche Forschungsgemeinschaft (CRC873,project A3)

  • Joachim Wittbrodt

Deutsche Forschungsgemeinschaft (FOR2509 P10,WI 1824/9-1)

  • Joachim Wittbrodt

Deutsche Forschungsgemeinschaft (CRC1118,project S03)

  • Marc Freichel

H2020 European Research Council (GA 294354-ManISteC)

  • Joachim Wittbrodt

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

Reviewing Editor

  1. Zacharias Kontarakis, ETH Zurich, Germany

Publication history

  1. Received: May 22, 2021
  2. Preprint posted: May 28, 2021 (view preprint)
  3. Accepted: March 15, 2022
  4. Accepted Manuscript published: March 25, 2022 (version 1)
  5. Accepted Manuscript updated: March 29, 2022 (version 2)
  6. Version of Record published: May 4, 2022 (version 3)

Copyright

© 2022, Thumberger 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. Thomas Thumberger
  2. Tinatini Tavhelidse-Suck
  3. Jose Arturo Gutierrez-Triana
  4. Alex Cornean
  5. Rebekka Medert
  6. Bettina Welz
  7. Marc Freichel
  8. Joachim Wittbrodt
(2022)
Boosting targeted genome editing using the hei-tag
eLife 11:e70558.
https://doi.org/10.7554/eLife.70558

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