Li Guoling, Dong Xue, Luo Jiamin, Yuan Tanglong, Li Tong, Zhao Guoli, Zhang Hainan, Zhou Jingxing, Zeng Zhenhai, Cui Shuna, Wang Haoqiang, Wang Yin, Yu Yuyang, Yuan Yuan, Zuo Erwei, Xu Chunlong, Huang Jinhai, Zhou Yingsi
HuidaGene Therapeutics Co., Ltd., Shanghai, 200131, China.
Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China.
Nat Commun. 2024 Sep 16;15(1):8090. doi: 10.1038/s41467-024-52485-1.
The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.
用于胞嘧啶碱基编辑器（CBE）的工程化TadA变体具有独特优势，与依赖天然脱氨酶的胞嘧啶碱基编辑器相比，其尺寸更小且脱靶效应更少。然而，当前的TadA变体在具有特定基序序列的DNA中表现出对碱基编辑的偏好，并具有双重脱氨酶活性，作用于相邻位置的胞嘧啶和腺苷，限制了它们的应用范围。为了解决这些问题，我们采用TadA直系同源物筛选和多序列比对（MSA）指导的蛋白质工程技术，创建了一种不受基序和腺苷脱氨酶活性限制的高效胞嘧啶碱基编辑器（aTdCBE）。值得注意的是，将aTdCBE递送至杜氏肌营养不良症（DMD）小鼠的人源化小鼠模型中，可实现强大的外显子55跳跃并恢复肌营养不良蛋白的表达。我们在工程化用于胞嘧啶编辑的TadA直系同源物方面的进展丰富了用于基因编辑治疗和其他潜在应用的碱基编辑工具包。
