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.

中文翻译：

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Cas9 介导的腺苷瞬时报告基因能够富集 ABE 靶向细胞


腺嘌呤碱基编辑器 (ABE) 可以实现单核苷酸修饰，无需传统的基于 CRIPSR/Cas9 的方法诱导的双链 DNA 断裂 (DSB)。然而，大多数采用 ABE 的方法都需要低效的下游技术来识别大量被操纵细胞中所需的靶向突变。在本研究中，我们开发了一种基于荧光的方法，称为“Cas9介导的腺苷瞬时报告基因编辑富集”（CasMAs-TREE；本文缩写为XMAS-TREE），以促进碱基编辑细胞群的实时识别。为了建立能够实时检测细胞内 ABE 活性的基于荧光的检测方法，我们设计了一种编码 mCherry 荧光蛋白的构建体，后跟绿色荧光蛋白 (GFP) 编码序列之前的终止密码子 (TGA)，从而允许将密码子从 A 转换为“TGG”后，进行翻译通读和 GFP 表达。在几个独立位点，我们证明 XMAS-TREE 可用于高效纯化目标细胞。此外，我们证明 XMAS-TREE 可以在多重编辑策略的背景下使用，以同时修改多个基因组位点。此外，我们利用 XMAS-TREE 来有效编辑人类多能干细胞 (hPSC)，这是一种传统上对基因修饰具有抵抗力的细胞类型。此外，我们利用 XMAS-TREE 在使用成熟的基于转染报告基因 (RoT) 的策略不可编辑的靶位点生成克隆同基因 hPSC。我们建立了一种检测细胞内腺苷碱基编辑活性的方法，该方法通过富集编辑的细胞来提高多个基因组位置的编辑效率。 未来，XMAS-TREE将极大加速ABE在生物医学研究中的应用。