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使用高保真DddA来源的碱基编辑器精确编辑线粒体DNA
2022-10-16 19:04

韩国基础科学研究所基因组工程中心Jin-Soo Kim团队近期取得重要工作进展,他们研究提出了使用高保真DddA来源的碱基编辑器精确编辑线粒体DNA。相关研究工作2022年10月13日在线发表于《自然—生物技术》杂志上。

研究人员设计了高保真DddA衍生的胞嘧啶碱基编辑器(HiFi-DdCBEs),通过在分割的DddAtox两半之间的界面用丙氨酸取代氨基酸残基,使脱靶活性最小化。产生的结构域如果没有与DNA上相邻位置的TALE蛋白结合,就不能形成功能性脱氨酶。全线粒体基因组测序表明,与传统的DdCBEs不同,HiFi-DdCBEs高效和精确,避免了附带的脱靶突变,可能会被用于治疗应用。传统的DdCBEs会在人类mtDNA中诱导数百个脱靶的C到T转换。

据介绍,细菌毒素DddA来源的胞嘧啶碱基编辑器(DdCBEs)由分割的DddAtox(一种双链DNA特异性胞嘧啶脱氨酶)、特定设计的TALE(转录激活样效应物)DNA结合蛋白和尿嘧啶糖基化酶抑制物(UGI)组成,可使人类细胞中的线粒体DNA(mtDNA)发生编辑,这可能为患者致病性mtDNA突变的治疗纠正铺平道路。DdCBEs的效用一直受到脱靶活性的限制,脱靶活性可能是由分割的DddAtox脱氨酶的自发组装决定的,与DNA结合的相互作用无关。

附:英文原文

Title: Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors

Author: Lee, Seonghyun, Lee, Hyunji, Baek, Gayoung, Kim, Jin-Soo

Issue&Volume: 2022-10-13

Abstract: Bacterial toxin DddA-derived cytosine base editors (DdCBEs)—composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor—enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.

DOI: 10.1038/s41587-022-01486-w

Source: https://www.nature.com/articles/s41587-022-01486-w

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:68.164
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex


本期文章:《自然—生物技术》:Online/在线发表

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