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    A transient reporter for editing enrichment (TREE) in human cells

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    Author
    Standage-Beier, Kylie
    Tekel, Stefan J
    Brookhouser, Nicholas
    Schwarz, Grace
    Nguyen, Toan
    Wang, Xiao
    Brafman, David A
    Affiliation
    Univ Arizona, Coll Med Phoenix, Grad Program Clin Translat Sci
    Issue Date
    2019-11-20
    
    Metadata
    Show full item record
    Publisher
    OXFORD UNIV PRESS
    Citation
    Kylie Standage-Beier, Stefan J Tekel, Nicholas Brookhouser, Grace Schwarz, Toan Nguyen, Xiao Wang, David A Brafman, A transient reporter for editing enrichment (TREE) in human cells, Nucleic Acids Research, Volume 47, Issue 19, 04 November 2019, Page e120, https://doi.org/10.1093/nar/gkz713
    Journal
    NUCLEIC ACIDS RESEARCH
    Rights
    Copyright © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
    Collection Information
    This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.
    Abstract
    Current approaches to identify cell populations that have been modified with deaminase base editing technologies are inefficient and rely on downstream sequencing techniques. In this study, we utilized a blue fluorescent protein (BFP) that converts to green fluorescent protein (GFP) upon a C-to-T substitution as an assay to report directly on base editing activity within a cell. Using this assay, we optimize various base editing transfection parameters and delivery strategies. Moreover, we utilize this assay in conjunction with flow cytometry to develop a transient reporter for editing enrichment (TREE) to efficiently purify base-edited cell populations. Compared to conventional cell enrichment strategies that employ reporters of transfection (RoT), TREE significantly improved the editing efficiency at multiple independent loci, with efficiencies approaching 80%. We also employed the BFP-to-GFP conversion assay to optimize base editor vector design in human pluripotent stem cells (hPSCs), a cell type that is resistant to genome editing and in which modification via base editors has not been previously reported. At last, using these optimized vectors in the context of TREE allowed for the highly efficient editing of hPSCs. We envision TREE as a readily adoptable method to facilitate base editing applications in synthetic biology, disease modeling, and regenerative medicine.
    Note
    Open access journal
    ISSN
    0305-1048
    PubMed ID
    31428784
    DOI
    10.1093/nar/gkz713
    Version
    Final published version
    Sponsors
    United States Department of Health & Human Services National Institutes of Health (NIH) - USA [R01GM121698, R21AG056706, R01GM106081, R01GM131405]; Arizona Biomedical Research Commission [ADHS16-162401]; International Foundation for Ethical Research Fellowship [R21AG056706]
    ae974a485f413a2113503eed53cd6c53
    10.1093/nar/gkz713
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