Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA.
Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY 10065, USA.
Nucleic Acids Res. 2020 Apr 6;48(6):2841-2852. doi: 10.1093/nar/gkaa124.
Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a 'Gene On' (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.
碱基编辑（BE）是一种强大的工具，可用于工程单核苷酸变体（SNVs），并已用于在细胞系、类器官和动物模型中创建靶向突变。最近新型 BE 酶的发展为基因组修饰提供了广泛的工具包；然而，鉴定和分离用于分析的编辑细胞一直具有挑战性。在这里，我们报告了一种“基因 ON”（GO）报告系统，该系统可原位高灵敏度和特异性地指示精确的胞嘧啶或腺嘌呤碱基编辑。我们使用可激活 GFP 来测试 GO，并使用它来测量一系列新型 BE 变体的动力学、效率和 PAM 特异性。此外，GO 具有灵活性，可以轻松适应诱导表达众多遗传编码标记物、抗生素抗性基因或酶，如 Cre 重组酶。有了这些工具，GO 可以用于在人类和小鼠细胞系和类器官中将 BE 事件与细胞内酶活性在内在基因组位置上进行功能链接。因此，GO 为在生物医学研究中实施 CRISPR BE 提供了一种强大的方法，提高了其实用性和可行性。
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