Abstract
Background: Adenine base editors (ABE) enable single nucleotide modifications without the need for double-stranded DNA breaks (DSBs) induced by conventional CRIPSR/Cas9-based approaches. However, most approaches that employ ABEs require inefficient downstream technologies to identify desired targeted mutations within large populations of manipulated cells. In this study, we developed a fluorescence-based method, named “Cas9-mediated adenosine transient reporter for editing enrichment” (CasMAs-TREE; herein abbreviated XMAS-TREE), to facilitate the real-time identification of base-edited cell populations. Results: To establish a fluorescent-based assay able to detect ABE activity within a cell in real time, we designed a construct encoding a mCherry fluorescent protein followed by a stop codon (TGA) preceding the coding sequence for a green fluorescent protein (GFP), allowing translational readthrough and expression of GFP after A-to-G conversion of the codon to “TGG.” At several independent loci, we demonstrate that XMAS-TREE can be used for the highly efficient purification of targeted cells. Moreover, we demonstrate that XMAS-TREE can be employed in the context of multiplexed editing strategies to simultaneous modify several genomic loci. In addition, we employ XMAS-TREE to efficiently edit human pluripotent stem cells (hPSCs), a cell type traditionally resistant to genetic modification. Furthermore, we utilize XMAS-TREE to generate clonal isogenic hPSCs at target sites not editable using well-established reporter of transfection (RoT)-based strategies. Conclusion: We established a method to detect adenosine base-editing activity within a cell, which increases the efficiency of editing at multiple genomic locations through an enrichment of edited cells. In the future, XMAS-TREE will greatly accelerate the application of ABEs in biomedical research.
Original language | English (US) |
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Article number | 193 |
Journal | BMC Biology |
Volume | 18 |
Issue number | 1 |
DOIs | |
State | Published - Dec 2020 |
Keywords
- Base editor
- CRISPR
- Genome modification
- Human pluripotent stem cells
- Multiplexing
ASJC Scopus subject areas
- Biotechnology
- Structural Biology
- Ecology, Evolution, Behavior and Systematics
- Physiology
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)
- Plant Science
- Developmental Biology
- Cell Biology
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Additional file 13 of A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells
Nguyen, T. (Contributor), Tekel, S. J. (Contributor), Brookhouser, N. (Contributor), Brafman, D. (Contributor), Standage-Beier, K. (Contributor) & Wang, X. (Contributor), figshare Academic Research System, Jan 1 2020
DOI: 10.6084/m9.figshare.13467893.v1, https://doi.org/10.6084%2Fm9.figshare.13467893.v1
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Additional file 3 of A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells
Standage-Beier, K. (Contributor), Brafman, D. (Contributor), Nguyen, T. (Contributor), Tekel, S. J. (Contributor), Brookhouser, N. (Contributor) & Wang, X. (Contributor), figshare Academic Research System, Jan 1 2020
DOI: 10.6084/m9.figshare.13467919.v1, https://doi.org/10.6084%2Fm9.figshare.13467919.v1
Dataset
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A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells
Brookhouser, N. (Creator), Nguyen, T. (Creator), Tekel, S. J. (Creator), Standage-Beier, K. (Creator), Wang, X. (Creator) & Brafman, D. (Creator), figshare Academic Research System, 2020
DOI: 10.6084/m9.figshare.c.5246415.v1, https://springernature.figshare.com/collections/A_Cas9-mediated_adenosine_transient_reporter_enables_enrichment_of_ABE-targeted_cells/5246415/1
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