Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting CE_CBE RNP complexes efficiently target cytosines in TC dinucleotides with minimal off-target or bystander mutations. Delivery of additional uracil glycosylase inhibitor protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation. A single electroporation was sufficient to effectively edit the therapeutically relevant locus BCL11A for sickle cell disease in hematopoietic stem and progenitor cells in a dose-dependent manner without cellular toxicity. Significantly, these CE_CBE RNPs permitted highly efficient editing and engraftment of transplanted cells in mice. Thus, our designed CBE proteins provide promising reagents for RNP-based editing at disease-related sites.

中文翻译：

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将 Cas 包埋的胞嘧啶碱基编辑器作为核糖核蛋白复合物直接递送，用于高效、准确地编辑临床相关靶标


最近，胞嘧啶碱基编辑器 （CBE） 已成为一种很有前途的治疗工具，用于特异性编辑单核苷酸变体和破坏与疾病相关的特定基因。尽管有这一承诺，但目前可用的 CBE 仍存在脱靶和旁观者编辑活动的重大责任，部分原因是它们的交付机制，导致其潜在应用受到限制。在这项研究中，我们设计了优化的、可溶的和稳定的 Cas-包埋 CBE （CE_CBEs），它整合了几项最新的进展，这些进展被有效地配制为作为核糖核蛋白 （RNP） 复合物直接递送到细胞中。我们得到的 CE_CBE RNP 复合物可有效靶向 TC 二核苷酸中的胞嘧啶，脱靶或旁观者突变极少。以反式方式递送额外的尿嘧啶糖基化酶抑制剂蛋白以剂量依赖性方式进一步提高了 C-to-T 编辑效率和靶标纯度，从而最大限度地减少了插入缺失的形成。单次电穿孔足以以剂量依赖性方式有效编辑造血干细胞和祖细胞中镰状细胞病的治疗相关基因座 BCL11A，而无细胞毒性。值得注意的是，这些CE_CBE RNP 允许在小鼠中高效编辑和植入移植细胞。因此，我们设计的 CBE 蛋白为疾病相关位点基于 RNP 的编辑提供了有前途的试剂。