当前位置:科学网首页 > 小柯机器人 >详情
研究实现基于CRISPR的高效特异性胞嘧啶碱基编辑
作者:小柯机器人 发布时间:2022/11/13 14:07:54

华东师范大学Dali Li,Mingyao Liu和北京大学Chengqi Yi共同合作近期取得重要工作进展,他们研究利用重组腺嘌呤脱氨酶TadA-8e,实现基于CRISPR的高效特异性胞嘧啶碱基编辑。相关研究成果2022年11月10日在线发表于《自然—生物技术》杂志上。

为了探索非天然的胞嘧啶脱氨酶,研究人员重新利用腺嘌呤脱氨酶TadA-8e进行胞嘧啶转化。TadA-8e中N46L变体的引入消除了其腺嘌呤脱氨酶活性,并产生了TadA-8来源的C-to-G碱基编辑器(Td-CGBE),能够高效精确地进行C·G-to-G·C编辑。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,获得了一系列Td-CBE,其活性与BE4max相似,或与其他报道的准确CBE相比具有更高的精密度。Td-CGBE/Td-CBE显示出非常低的indel效应和背景水平的Cas9依赖或Cas9独立的DNA/RNA脱靶编辑。

此外,Td-CGBE/Td-CBEs在细胞或小鼠胚胎同聚胞嘧啶位点产生准确编辑方面更有效率,表明它们在基因治疗和其他应用方面的准确性和安全性。

据介绍,胞嘧啶碱基编辑器(CBE)能有效地产生精确的C·G至T·A碱基转换,但激活诱导的胞苷脱氨酶/载脂蛋白B mRNA编辑酶催化多肽样蛋白(AID/APOBEC)家族脱氨酶组分,会诱导相当大的脱靶效应并且产生碱基的插入和缺失(indel)。

附:英文原文

Title: Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing

Author: Chen, Liang, Zhu, Biyun, Ru, Gaomeng, Meng, Haowei, Yan, Yongchang, Hong, Mengjia, Zhang, Dan, Luan, Changming, Zhang, Shun, Wu, Hao, Gao, Hongyi, Bai, Sijia, Li, Changqing, Ding, Ruoyi, Xue, Niannian, Lei, Zhixin, Chen, Yuting, Guan, Yuting, Siwko, Stefan, Cheng, Yiyun, Song, Gaojie, Wang, Liren, Yi, Chengqi, Liu, Mingyao, Li, Dali

Issue&Volume: 2022-11-10

Abstract: 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.

DOI: 10.1038/s41587-022-01532-7

Source: https://www.nature.com/articles/s41587-022-01532-7

期刊信息

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex