5' modifications improve potency and efficacy of DNA donors for precision genome editing
- University of Massachusetts Medical School, United States
- UMass medical School, United States
- UMass Medical School, United States
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
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Data from: 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome EditingDryad Digital Repository, doi.10.5061/dryad.f7m0cfxwr.
Article and author information
Author details
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
- Jessica K Tyler, Weill Cornell Medicine, United States
Publication history
- Received: July 29, 2021
- Accepted: September 9, 2021
- Accepted Manuscript published: October 19, 2021 (version 1)
- 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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5' modifications improve potency and efficacy of DNA donors for precision genome editing
eLife 10:e72216.
https://doi.org/10.7554/eLife.72216
Further reading
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Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).
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