A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells

• Published in: BMC biology 2020-12, Vol.18 (1), p.193-193, Article 193
• Main Authors: Brookhouser, Nicholas, Nguyen, Toan, Tekel, Stefan J, Standage-Beier, Kylie, Wang, Xiao, Brafman, David A
• Format: Article
• Language: English
• Subjects: Adenine; Adenine - chemistry; Adenosine; Adenosine - genetics; Adenosine - metabolism; Base Composition; Base editor; Cell cycle; Cell division; Conversion; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; Deoxyribonucleic acid; DNA; DNA damage; Editing; Editors; Efficiency; Enrichment; Enzymes; Flow cytometry; Fluorescence; Gene Editing - methods; Gene loci; Gene targeting; Genes, Reporter; Genetic modification; Genome modification; Genomes; Green fluorescent protein; Human pluripotent stem cells; Humans; Identification and classification; Luminescent Proteins - chemistry; Methods; Microscopy; Multiplexing; Mutation; Nucleotides; Pluripotency; Pluripotent Stem Cells - metabolism; Populations; Proteins; Real time; Red Fluorescent Protein; Rot; Single-Cell Analysis; Stem cells; Stop codon; Transfection
• ISSN: 1741-7007; 1741-7007
• DOI: 10.1186/s12915-020-00929-7

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. 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. 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.