College of Animal Sciences, Jilin University, Changchun 130062, China.
Chongqing Research Institute, Jilin University, Chongqing 401123, China.
Nucleic Acids Res. 2024 Oct 14;52(18):11188-11202. doi: 10.1093/nar/gkae800.
Cytidine base editors (CBEs) hold significant potential in genetic disease treatment and in breeding superior traits into animals. However, their large protein sizes limit their delivery by adeno-associated virus (AAV), given its packing capacity of <4.7 kb. To overcome this, we employed a web-based fast generic discovery (WFG) strategy, identifying several small ssDNA deaminases (Sdds) and constructing multiple Sdd-CBE 1.0 versions. SflSdd-CBE 1.0 demonstrated high C-to-T editing efficiency, comparable to AncBE4max, while SviSdd-CBE 1.0 exhibited moderate C-to-T editing efficiency with a narrow editing window (C3 to C5). Utilizing AlphaFold2, we devised a one-step miniaturization strategy, reducing the size of Sdds while preserving their efficiency. Notably, we administered AAV8 expressing PCSK9 targeted sgRNA and SflSdd-CBEs (nSaCas9) 2.0 into mice, leading to gene-editing events (with editing efficiency up to 15%) and reduced serum cholesterol levels, underscoring the potential of Sdds in gene therapy. These findings offer new single-stranded editing tools for the treatment of rare genetic diseases.
胞嘧啶碱基编辑器（CBEs）在遗传疾病治疗和将优异性状培育到动物中具有重要潜力。然而，由于腺相关病毒（AAV）的包装能力<4.7 kb，其较大的蛋白尺寸限制了它们的传递。为了克服这一限制，我们采用了基于网络的快速通用发现（WFG）策略，鉴定了几种小的 ssDNA 脱氨酶（Sdds），并构建了多个 Sdd-CBE 1.0 版本。SflSdd-CBE 1.0 表现出高的 C 到 T 的编辑效率，与 AncBE4max 相当，而 SviSdd-CBE 1.0 则表现出中等的 C 到 T 的编辑效率和狭窄的编辑窗口（C3 到 C5）。利用 AlphaFold2，我们设计了一种一步缩小尺寸的策略，在保持效率的同时缩小 Sdds 的尺寸。值得注意的是，我们向小鼠体内注射表达 PCSK9 靶向 sgRNA 和 SflSdd-CBEs（nSaCas9）2.0 的 AAV8，导致基因编辑事件（编辑效率高达 15%）和血清胆固醇水平降低，这突出了 Sdds 在基因治疗中的潜力。这些发现为治疗罕见遗传疾病提供了新的单链编辑工具。
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