A transient reporter for editing enrichment (TREE) in human cells
Tekel, Stefan J
Brafman, David A
AffiliationUniv Arizona, Coll Med Phoenix, Grad Program Clin Translat Sci
MetadataShow full item record
PublisherOXFORD UNIV PRESS
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/gkz713
JournalNUCLEIC ACIDS RESEARCH
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/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Collection InformationThis 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 firstname.lastname@example.org.
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.
NoteOpen access journal
VersionFinal published version
SponsorsUnited 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]
- A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells.
- Authors: Brookhouser N, Nguyen T, Tekel SJ, Standage-Beier K, Wang X, Brafman DA
- Issue date: 2020 Dec 14
- BIG-TREE: Base-Edited Isogenic hPSC Line Generation Using a Transient Reporter for Editing Enrichment.
- Authors: Brookhouser N, Tekel SJ, Standage-Beier K, Nguyen T, Schwarz G, Wang X, Brafman DA
- Issue date: 2020 Feb 11
- Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.
- Authors: Yumlu S, Stumm J, Bashir S, Dreyer AK, Lisowski P, Danner E, Kühn R
- Issue date: 2017 May 15
- Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs.
- Authors: Okamoto S, Amaishi Y, Maki I, Enoki T, Mineno J
- Issue date: 2019 Mar 18
- CRISPR Base Editing in Induced Pluripotent Stem Cells.
- Authors: Chang YJ, Xu CL, Cui X, Bassuk AG, Mahajan VB, Tsai YT, Tsang SH
- Issue date: 2019