A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells.
BMC Biol
; 18(1): 193, 2020 12 14.
Article
em En
| MEDLINE
| ID: mdl-33317513
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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Adenosina
/
Genes Reporter
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Células-Tronco Pluripotentes
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Edição de Genes
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Proteína 9 Associada à CRISPR
Limite:
Humans
Idioma:
En
Revista:
BMC Biol
Assunto da revista:
BIOLOGIA
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos