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

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.

Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing

“…In order to compare the editing efficiency enriched by CBE-ARSR-1 × ACG and CBE-ARSR-2 × ACG, we constructed five sgRNA vectors targeting five genomic loci (EMX1, WRNIP1, APOE, Site1, and Site2). The information of Site1 and Site2 from referencess ( 26 , 27 ). Cotransfected HEK293T cells with YE1-BE3-FNLS, sgRNA vector, and CBE-ARSR-1 × ACG or CBE-ARSR-2 × ACG.…”

“…Some of the fluorescence-based reporters were previously designed based on the assumption that a stop codon TAG/TGA can be converted to TGG by ABE, so that a fluorescent gene downstream can be expressed and used for evaluating the ABE activity ( 26 ). To develop a universal surrogate reporter that can be used to simultaneously enrich the cells edited by either ABE or CBE, we constructed a new reporter vector ACBE-ARSR on the basis of CBE-ARSR-1 × ACG reporter construct for evaluating both adenosine base editing and cytosine base editing ( Fig.…”

“…Recently, several fluorescence-based reporters have been developed to report CBE or ABE activities ( 26 , 27 , 33 , 34 , 35 , 36 , 37 , 38 ). The codon CAC at the 66th position of BFP, when modified to ‘TAC’ or ‘TAT’ by CBE, can result in the amino acid change from histidine to tyrosine, making the fluorescence shift from BFP to GFP.…”

Fast and Efficient Generation of Isogenic Induced Pluripotent Stem Cell Lines Using Adenine Base Editing

“…15 BE has been successfully used in vitro in many different cell culture and animal models, [16][17][18][19][20] but only very recently for generation of isogenic iPSC lines. [21][22][23][24][25]…”

CRISPR in cancer biology and therapy

“…We expect to see more active variants and adaptations appear in the com ing years. Until then, the use of BE and PE reporters [40][41][42][43] is an effective approach to enrich target editing and streamline the creation of engineered cells and animals for cancer gene discovery.…”