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

Kylie Standage-Beier, Stefan J. Tekel, Nicholas Brookhouser, Grace Schwarz, Toan Nguyen, Xiao Wang, David A. Brafman

Research output: Contribution to journalArticle

5 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)e120
JournalNucleic acids research
Volume47
Issue number19
DOIs
StatePublished - Nov 4 2019

ASJC Scopus subject areas

  • Genetics

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    Standage-Beier, K., Tekel, S. J., Brookhouser, N., Schwarz, G., Nguyen, T., Wang, X., & Brafman, D. A. (2019). A transient reporter for editing enrichment (TREE) in human cells. Nucleic acids research, 47(19), e120. https://doi.org/10.1093/nar/gkz713