Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an adenine BE (which converts A·T base pairs to G·C) and a cytosine BE (which converts C·G base pairs to T·A), is not feasible because guide RNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here we engineer both adenine BEs and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer–coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs. We generate four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies. The addition of a fluorescent enrichment strategy increases co-occurring edit rates up to 24.8% in human cells. These systems are compatible with expanded protospacer adjacent motif and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared with their parental systems and can model disease-relevant point mutation combinations.

碱基编辑器 (BE) 可实现点突变的高效、可编程安装，同时避免使用双链断裂。同时应用两种或多种不同的 BE，例如腺嘌呤 BE（将 A·T 碱基对转换为 G·C）和胞嘧啶 BE（将 C·G 碱基对转换为 T·A），是不可行的，因为RNA 串扰会导致非正交编辑，所有 BE 都会修改所有目标基因座。在这里，我们设计了腺嘌呤 BE 和胞嘧啶 BE，可以通过使用 RNA 适体-外壳蛋白系统将 DNA 修饰酶直接招募到引导 RNA 上进行正交多重化。我们生成了四个多重正交 BE 系统，无需富集或选择策略，即可在同一 DNA 链中实现高达 7.1% 的精确共现编辑率。添加荧光富集策略可将人类细胞中的共发生编辑率提高至 24.8%。这些系统与扩展的原型间隔子相邻基序和高保真 Cas9 变体兼容，在多种细胞类型中功能良好，与其亲本系统相比具有相同或降低的脱靶倾向，并且可以模拟与疾病相关的点突变组合。