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dc.contributor.authorStandage-Beier, Kylie
dc.contributor.authorTekel, Stefan J
dc.contributor.authorBrookhouser, Nicholas
dc.contributor.authorSchwarz, Grace
dc.contributor.authorNguyen, Toan
dc.contributor.authorWang, Xiao
dc.contributor.authorBrafman, David A
dc.date.accessioned2019-12-13T00:28:05Z
dc.date.available2019-12-13T00:28:05Z
dc.date.issued2019-11-20
dc.identifier.citationKylie 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/gkz713en_US
dc.identifier.issn0305-1048
dc.identifier.pmid31428784
dc.identifier.doi10.1093/nar/gkz713
dc.identifier.urihttp://hdl.handle.net/10150/636359
dc.description.abstractCurrent 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.en_US
dc.description.sponsorshipUnited 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]en_US
dc.language.isoenen_US
dc.publisherOXFORD UNIV PRESSen_US
dc.rightsCopyright © 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/).en_US
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.titleA transient reporter for editing enrichment (TREE) in human cellsen_US
dc.typeArticleen_US
dc.contributor.departmentUniv Arizona, Coll Med Phoenix, Grad Program Clin Translat Scien_US
dc.identifier.journalNUCLEIC ACIDS RESEARCHen_US
dc.description.noteOpen access journalen_US
dc.description.collectioninformationThis 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.en_US
dc.eprint.versionFinal published versionen_US
dc.source.journaltitleNucleic acids research
refterms.dateFOA2019-12-13T00:28:05Z


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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/).
Except where otherwise noted, this item's license is described as 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/).