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

BMC Biology

David A. Brafman¹  Stefan J. Tekel¹  Toan Nguyen¹  Xiao Wang¹  Nicholas Brookhouser²  Kylie Standage-Beier³ 

[1] School of Biological and Health Systems Engineering, Arizona State University, 501 E. Tyler Mall, ECG 334A, 85287, Tempe, AZ, USA;School of Biological and Health Systems Engineering, Arizona State University, 501 E. Tyler Mall, ECG 334A, 85287, Tempe, AZ, USA;Graduate Program in Clinical Translational Sciences, University of Arizona College of Medicine-Phoenix, 85004, Phoenix, AZ, USA;School of Biological and Health Systems Engineering, Arizona State University, 501 E. Tyler Mall, ECG 334A, 85287, Tempe, AZ, USA;Molecular and Cellular Biology Graduate Program, Arizona State University, 85287, Tempe, AZ, USA;

关键词: CRISPR;    Genome modification;    Base editor;    Human pluripotent stem cells;    Multiplexing;   

DOI  :  10.1186/s12915-020-00929-7

来源: Springer

【 摘 要 】

BackgroundAdenine 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.ResultsTo 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.ConclusionWe 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.

【 授权许可】

CC BY