College of Animal Sciences, Jilin University, Changchun 130062, China.
College of Animal Sciences, Jilin University, Changchun 130062, China; Chongqing Research Institute, Jilin University, Chongqing 401123, China; Chongqing Jitang Biotechnology Research Institute, Chongqing 401123, China.
Int J Biol Macromol. 2023 Dec 31;253(Pt 7):127418. doi: 10.1016/j.ijbiomac.2023.127418. Epub 2023 Oct 16.
Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.
腺嘌呤碱基编辑器能够在基因组 DNA 中实现靶向的 A 到 G 转换，在治疗应用中具有巨大的潜力。然而，目前使用的腺嘌呤碱基编辑器在基因治疗中受到宽编辑窗口和脱靶效应的限制。在这里，我们报告了人类 e18 蛋白，一种 RING 型 E3 泛素连接酶变体，与腺嘌呤碱基编辑器融合，可以通过减少碱基编辑器蛋白的丰度，显著提高精度并缩小编辑窗口，与 ABEmax 和 ABE8e 相比。作为概念验证，ABEmax-e18 和 ABE8e-e18 显著降低了 Cas9 依赖性和 Cas9 非依赖性脱靶效应，比传统的腺嘌呤碱基编辑器更明显。此外，我们利用 ABEmax-e18 建立了并指畸形小鼠模型，并在 HepG2 细胞中实现了人 PCSK9 基因座的精确碱基转换，这表明其在基因治疗中的潜力。此外，碱基编辑器的截断版本 - RING（ABEmax-RING 或 AncBE4max-RING），将 e18 蛋白 RING 结构域的 63 个氨基酸融合到 ABEmax 或 AncBE4max 的 C 端，与 ABEmax-e18 或 AncBE4max-e18 相比，表现出相似的效果。总之，与碱基编辑器融合的 e18 或 RING 蛋白增强了基因修饰的精确工具包，并且可能与各种碱基编辑工具很好地结合，在未来具有更广泛的精确基因治疗应用前景。
