Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies—for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1–3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.

基因组编辑改变了生命科学，并在治疗遗传疾病方面具有令人兴奋的前景。我们的实验室开发了碱基编辑技术，以实现精确、高效的基因组编辑，同时最大限度地减少与双链 DNA 断裂相关的不良副产物和毒性。腺嘌呤和胞嘧啶碱基编辑器分别介导靶向A•T至G•C或C•G至T•A碱基对变化，理论上可以解决大多数人类疾病相关的单核苷酸多态性。目前的碱基编辑器可以实现很高的编辑效率，例如，在培养的哺乳动物细胞中接近 100%，在体内成年小鼠神经元中接近 70%。自最初的描述以来，已经开发出大量具有不同的在靶和脱靶编辑特征的碱基编辑器变体。在这里，我们描述了在培养的哺乳动物细胞中使用碱基编辑的协议。我们提供选择目标位点、适当的碱基编辑器变体和交付策略的指南，以最适合所需的应用。我们进一步描述了 HEK293T 细胞中的标准碱基编辑实验，以及使用 CRISPResso2 对碱基编辑结果的计算分析。从目标 DNA 位点选择开始，哺乳动物细胞中的碱基编辑实验通常可以在 1-3 周内完成，并且只需要标准分子生物学技术和现成的质粒构建体。