Laboratory of Genomic and Precision Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
Laboratory of Genomic and Precision Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China; Department of Pediatric Laboratory, Affiliated Children's Hospital of Jiangnan University (Wuxi Children's Hospital), Wuxi, Jiangsu, China.
Cell Rep. 2024 Mar 26;43(3):113878. doi: 10.1016/j.celrep.2024.113878. Epub 2024 Mar 2.
Cytidine deaminase defines the properties of cytosine base editors (CBEs) for C-to-T conversion. Replacing the cytidine deaminase rat APOBEC1 (rA1) in CBEs with a human APOBEC3A (hA3A) improves CBE properties. However, the potential CBE application of macaque A3A orthologs remains undetermined. Our current study develops and evaluates engineered CBEs based on Macaca fascicularis A3A (mA3A). Here, we demonstrate that BE4-mA3A and its RNA-editing-derived variants exhibit improved CBE properties, except for DNA off-target activity, compared to BE3-rA1 and BE4-rA1. Unexpectedly, deleting Ser-Val-Arg (SVR) in BE4-mA3A dramatically reduces DNA and RNA off-target activities and improves editing accuracy, with on-target efficiency unaffected. In contrast, a chimeric BE4-hA3A-SVR shows editing efficiency increased by about 50%, with other properties unaffected. Our findings demonstrate that mA3A-based CBEs could provide prototype options with advantages over rA1- and hA3A-based CBEs for further optimization, highlighting the importance of the SVR motif in defining CBE intrinsic properties.
胞嘧啶脱氨酶定义了胞嘧啶碱基编辑器（CBEs）将 C 转换为 T 的特性。用人类 APOBEC3A（hA3A）替代 CBEs 中的大鼠 APOBEC1（rA1）可改善 CBE 特性。然而，猕猴 A3A 同源物的潜在 CBE 应用仍未确定。我们目前的研究基于恒河猴 A3A（mA3A）开发和评估了工程化的 CBE。在这里，我们证明与 BE3-rA1 和 BE4-rA1 相比，BE4-mA3A 及其 RNA 编辑衍生变体具有更好的 CBE 特性，除了 DNA 脱靶活性。出乎意料的是，删除 BE4-mA3A 中的丝氨酸-缬氨酸-精氨酸（SVR）会显著降低 DNA 和 RNA 脱靶活性并提高编辑准确性，而不会影响靶标效率。相比之下，嵌合 BE4-hA3A-SVR 的编辑效率提高了约 50%，其他特性不受影响。我们的研究结果表明，基于 mA3A 的 CBE 可以提供优于基于 rA1 和 hA3A 的 CBE 的原型选择，以进一步优化，突出了 SVR 基序在定义 CBE 内在特性方面的重要性。
Zotero 插件