Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
Bioray Laboratories, Shanghai, China.
Nat Cell Biol. 2020 Jun;22(6):740-750. doi: 10.1038/s41556-020-0518-8. Epub 2020 May 11.
Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.
胞嘧啶碱基编辑器是一种强大的遗传工具，可在特定基因组位点催化胞嘧啶向胸腺嘧啶的转化，进一步提高编辑范围和效率对于它们的更广泛应用至关重要。通过在 Cas9 切口酶和脱氨酶之间插入来自 Rad51 蛋白的非序列特异性单链 DNA 结合结构域，生成了一系列超胞嘧啶碱基编辑器，其活性大大提高，并且在细胞系和小鼠胚胎中朝着邻近基序的编辑窗口扩大。此外，与 eA3A-BE4max 相比，hyeA3A-BE4max 选择性地在 TC 基序中催化胞嘧啶转化，具有更宽的编辑范围和更高的活性（高达 257 倍）。此外，hyeA3A-BE4max 特异性地产生 C 到 T 的转换，而不会在血红蛋白γ基因启动子中诱导旁观者突变，从而模拟自然发生的遗传变异以改善β-地中海贫血症，这表明改进的碱基编辑器具有治疗潜力。
