Beam Therapeutics, Cambridge, MA, USA.
CRISPR J. 2021 Apr;4(2):169-177. doi: 10.1089/crispr.2020.0144.
Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.
碱基编辑器是脱氨酶和 CRISPR-Cas 核糖核蛋白的融合体，允许在没有双链 DNA 断裂中间体的情况下可编程地安装转换突变。潜在碱基编辑靶标的广度通常受到 Cas9 前导间隔相邻基序的适当位置的要求的限制。为了解决这个问题，我们使用 Cas9 和 TadA 的结构设计了一组嵌入式碱基编辑器 (IBE)，其中脱氨酶结构域位于 Cas9 内部。这些 IBE 中的几个表现出移位的编辑窗口和更高的编辑效率，使我们能够以降低的 DNA 和 RNA 脱靶编辑频率编辑规范编辑窗口之外的靶标。最后，我们表明 IBE 能够使患者来源的造血干细胞中的致病镰状细胞血红蛋白等位基因转化为天然存在的 HbG-Makassar 变体。
