Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when multiple Cs are present in the ~ 5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities. We used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3–58.0% and 54.5–92.2% with excellent CC context-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83–100% and few bystander mutations in Founder (F0) rabbits at Tyr loci. These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.

中文翻译：

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使用工程化的人类APOBEC3G-nCas9融合体，具有CC上下文特异性的精确碱基编辑。

胞嘧啶碱基编辑器（CBE）由与Cas9切口酶（nCas9）融合的胞苷脱氨酶组成，可在多种生物中实现有效的C-T转化。但是，当胞苷脱氨酶的〜5核苷酸活性窗口中存在多个C时，当前的碱基编辑者可能会引起不需要的旁观者C到T转换，这会对它们的准确性产生负面影响。在这里，我们开发了一个新的基本编辑器，该编辑器可以大大减少不必要的旁观者活动。我们使用具有优先胞嘧啶脱氨酶活性的工程化人APOBEC3G（eA3G）C末端催化结构域，在具有CCC> CCC> CC（其中优先脱氨基C带有下划线的层次结构）的基序中，开发了具有独特CC上下文的eA3G-BE -特异性和减少旁观者突变的产生。目标编辑效率为18.3–58.0％和54.5–92。在人细胞和兔胚胎中分别产生了2％的具有优异CC上下文特异性的蛋白。此外，通过将hA3G与工程化的SpCas9-NG变体相结合，可以实现可以进一步识别宽松的NG PAM的基础编辑器。A3G-BEs用于诱导准确的单碱基取代，从而导致在Tyr位点的Founder（F0）兔中无意义的突变，效率为83-100％，旁观者突变很少。这些新颖的基础编辑器具有更高的精度和CC上下文特异性，将扩展工具集，以便在生物体中进行精确的基因修饰。A3G-BEs用于诱导准确的单碱基取代，从而导致在Tyr位点的Founder（F0）兔中无意义的突变，效率为83-100％，旁观者突变很少。这些新颖的基础编辑器具有更高的精确度和CC上下文特异性，将扩展工具集，以便在生物体中进行精确的基因修饰。A3G-BEs用于诱导准确的单碱基取代，从而导致无义突变，效率为83–100％，在Tyr位点的Founder（F0）兔子中几乎没有旁观者突变。这些新颖的基础编辑器具有更高的精度和CC上下文特异性，将扩展工具集，以便在生物体中进行精确的基因修饰。