为实现高效且特异性的基于 CRISPR 的胞嘧啶碱基编辑,对腺嘌呤脱氨酶 TadA-8e 进行重新设计。
Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.
机构信息
Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
School of Life Sciences, Peking University, Beijing, China.
出版信息
Nat Biotechnol. 2023 May;41(5):663-672. doi: 10.1038/s41587-022-01532-7. Epub 2022 Nov 10.
Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.
胞嘧啶碱基编辑器(CBEs)能够高效地将 C·G 转化为 T·A 碱基对,但是激活诱导的胞嘧啶脱氨酶/载脂蛋白 B mRNA 编辑酶催化多肽样(AID/APOBEC)蛋白家族脱氨酶组件会引起显著的脱靶效应和插入缺失。为了探索非天然的胞嘧啶脱氨酶,我们将腺嘌呤脱氨酶 TadA-8e 重新用于胞嘧啶的转化。在 TadA-8e 中引入 N46L 变体可以消除其腺嘌呤脱氨酶活性,从而产生一种能够高效且精确地将 C·G 转化为 G·C 的 TadA-8e 衍生的 C 到 G 碱基编辑器(Td-CGBE)。通过与尿嘧啶糖基化酶抑制剂融合,并进一步引入其他变体,可以获得一系列 Td-CGBE,其活性与 BE4max 相当,或者与其他报道的精确 CBE 相比具有更高的精度。Td-CGBE/Td-CBEs 显示出非常低的插入缺失效应和 Cas9 依赖性或非依赖性的 DNA/RNA 脱靶编辑的背景水平。此外,Td-CGBE/Td-CBEs 在细胞或小鼠胚胎中多聚胞嘧啶位点产生精确编辑的效率更高,这表明它们在基因治疗和其他应用中具有准确性和安全性。