5' modifications improve potency and efficacy of DNA donors for precision genome editing

Abstract

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Data availability

All the sequencing data will be deposited to Dyrad and can be accessed at https://doi.org/10.5061/dryad.f7m0cfxwr

The following data sets were generated

Article and author information

Author details

  1. Krishna S Ghanta

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    Krishna S Ghanta, Co-inventor on patent applications related to this work (Application number: 16/384, 612 ).
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7502-3141
  2. Zexiang Chen

    UMass medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  3. Aamir Mir

    UMass Medical School, Worcester, United States
    Competing interests
    Aamir Mir, Co-inventor on patent application related to this work (Application number: 16/384, 612 ).
  4. Gregoriy A Dokshin

    UMass Medical School, Worcester, United States
    Competing interests
    Gregoriy A Dokshin, Co-inventor on patent applications related to this work (Application number: 16/384, 612 ).
  5. Pranathi M Krishnamurthy

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  6. Yeonsoo Yoon

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4302-9933
  7. Judith Gallant

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  8. Ping Xu

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  9. Xiao-Ou Zhang

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  10. Ahmet Rasit Ozturk

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  11. Masahiro Shin

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0231-894X
  12. Feston Idrizi

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8035-3951
  13. Pengpeng Liu

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  14. Hassan Gneid

    UMass Medical School, Worcester, United States
    Competing interests
    Hassan Gneid, Co-inventor on patent applications related to this work (Application number: 16/384, 612 ).
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9326-2023
  15. Alireza Edraki

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  16. Nathan D Lawson

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7788-9619
  17. Jaime A Rivera-Pérez

    UMass Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  18. Erik Sontheimer

    Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    Erik.Sontheimer@umassmed.edu
    Competing interests
    Erik Sontheimer, Co-inventor on patent applications related to this work (Application number: 16/384, 612 ).
  19. Jonathan K Watts

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    Jonathan.Watts@umassmed.edu
    Competing interests
    Jonathan K Watts, Co-inventor on patent applications related to this work (Application number: 16/384, 612 ).
  20. Craig C Mello

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    Craig.Mello@umassmed.edu
    Competing interests
    Craig C Mello, The authors (K.S.G, A.M, G.A.D, H.G, J.K.W, E.J.S and C.C.M) have a patent application pending related to the findings described (Application number: 16/384, 612 ). Craig C. Mello is a co-founder and Scientific Advisory Board member of CRISPR Therapeutics, and Erik J. Sontheimer is a cofounder and Scientific Advisory Board member of Intellia herapeutics..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9176-6551

Funding

Howard Hughes Medical Institute

  • Craig C Mello

Office of Extramural Research, National Institutes of Health (R37 GM058800-23)

  • Craig C Mello

National Center for Advancing Translational Sciences (UG3 TR002668)

  • Erik Sontheimer
  • Jonathan K Watts

National Center for Advancing Translational Sciences (UG3 TR002668)

  • Erik Sontheimer

National Heart, Lung, and Blood Institute (R35 HL140017)

  • Nathan D Lawson

NIH Office of the Director (R21 OD030004)

  • Nathan D Lawson

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

Ethics

Animal experimentation: Fish were maintained in accordance with the protocols set by the University of Massachusetts Medical School Institutional Animal Care and Use Committee. All the mouse experiments were conducted according the UMMS Institute Animal Care and Use Committee (IACUC).

Reviewing Editor

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

Publication history

  1. Received: July 29, 2021
  2. Accepted: September 9, 2021
  3. Accepted Manuscript published: October 19, 2021 (version 1)
  4. Version of Record published: November 4, 2021 (version 2)

Copyright

© 2021, Ghanta 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. Krishna S Ghanta
  2. Zexiang Chen
  3. Aamir Mir
  4. Gregoriy A Dokshin
  5. Pranathi M Krishnamurthy
  6. Yeonsoo Yoon
  7. Judith Gallant
  8. Ping Xu
  9. Xiao-Ou Zhang
  10. Ahmet Rasit Ozturk
  11. Masahiro Shin
  12. Feston Idrizi
  13. Pengpeng Liu
  14. Hassan Gneid
  15. Alireza Edraki
  16. Nathan D Lawson
  17. Jaime A Rivera-Pérez
  18. Erik Sontheimer
  19. Jonathan K Watts
  20. Craig C Mello
(2021)
5' modifications improve potency and efficacy of DNA donors for precision genome editing
eLife 10:e72216.
https://doi.org/10.7554/eLife.72216

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