Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. Recent studies report that BE3, the original CBE, induces a low frequency of genome-wide Cas9-independent off-target C•G-to-T•A mutation in mouse embryos and in rice. Here we develop multiple rapid, cost-effective methods to screen the propensity of different CBEs to induce Cas9-independent deamination in Escherichia coli and in human cells. We use these assays to identify CBEs with reduced Cas9-independent deamination and validate via whole-genome sequencing that YE1, a narrowed-window CBE variant, displays background levels of Cas9-independent off-target editing. We engineered YE1 variants that retain the substrate-targeting scope of high-activity CBEs while maintaining minimal Cas9-independent off-target editing. The suite of CBEs characterized and engineered in this study collectively offer ~10–100-fold lower average Cas9-independent off-target DNA editing while maintaining robust on-target editing at most positions targetable by canonical CBEs, and thus are especially promising for applications in which off-target editing must be minimized.

胞嘧啶碱基编辑器 (CBE) 可实现基因组 DNA 中的定向 C•G 到 T•A 转换。最近的研究报告称，BE3（最初的 CBE）在小鼠胚胎和水稻中诱导低频率的全基因组 Cas9 独立的脱靶 C•G 至 T•A 突变。在这里，我们开发了多种快速、经济有效的方法来筛选不同 CBE 在大肠杆菌和人类细胞中诱导不依赖于 Cas9 的脱氨作用的倾向。我们使用这些检测方法来鉴定与 Cas9 无关的脱氨基作用减少的 CBE，并通过全基因组测序验证 YE1（一种窄窗口 CBE 变体）显示出与 Cas9 无关的脱靶编辑的背景水平。我们设计了 YE1 变体，保留了高活性 CBE 的底物靶向范围，同时保持了最小程度的独立于 Cas9 的脱靶编辑。本研究中表征和设计的 CBE 套件共同提供了约 10-100 倍的平均 Cas9 独立脱靶 DNA 编辑能力，同时在规范 CBE 可靶向的大多数位置上保持稳健的靶向编辑，因此特别有前景。其中必须尽量减少脱靶编辑。