小柯机器人

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科学家开发出线粒体碱基编辑器
2020-07-11 13:32
来源：小柯机器人

美国哈佛大学刘如谦（David R. Liu）、华盛顿大学Joseph D. Mougous等研究人员合作开发出一种不依赖于CRISPR的线粒体碱基编辑器。相关论文于2020年7月8日在线发表在《自然》杂志上。

研究人员了一种细菌间毒素，并将其命名为DddA，它可以催化双链DNA的胞苷脱氨。研究人员设计了无毒的split-DddA半分子，并且其在结合靶标DNA之前没有活性。split-DddA半分子、转录激活因子样效应子阵列蛋白以及尿嘧啶糖基化酶抑制剂的融合产生了无RNA的DddA衍生型胞嘧啶碱基编辑器（DdCBE），其可催化人线粒体DNA中的C•G到T•A转化，并具有很高的目标特异性和产物纯度。研究人员使用DdCBE构建了人类细胞中与疾病相关的线粒体DNA突变模型，从而导致呼吸速率和氧化磷酸化的改变。不含CRISPR的DdCBE可以精确操纵线粒体DNA，而非消除线粒体DNA拷贝，这对线粒体疾病的研究和潜在疗法具有广泛意义。
 
据介绍，细菌毒素是一个巨大的生化多样性储备库，可以重新用于生物医学应用。此类蛋白质包括一类脱氨酶超家族的细菌间毒素，其成员已发现在基因编辑技术中的应用。由于以往报道的胞苷脱氨基酶在单链核酸上运行，因此它们在碱基编辑中需要解开双链DNA，例如通过CRISPR–Cas9系统。迄今为止，线粒体DNA中的碱基编辑受到向导RNA无法进入线粒体的阻碍。因此，对线粒体DNA的操作仅限于通过设计者核酸酶对线粒体基因组的靶向破坏。
 
附：英文原文

Title: A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing

Author: Beverly Y. Mok, Marcos H. de Moraes, Jun Zeng, Dustin E. Bosch, Anna V. Kotrys, Aditya Raguram, FoSheng Hsu, Matthew C. Radey, S. Brook Peterson, Vamsi K. Mootha, Joseph D. Mougous, David R. Liu

Issue&Volume: 2020-07-08

Abstract: Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse CG-to-TA conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

DOI: 10.1038/s41586-020-2477-4

Source: https://www.nature.com/articles/s41586-020-2477-4

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
官方网址：http://www.nature.com/
投稿链接：http://www.nature.com/authors/submit_manuscript.html


本期文章：《自然》：Online/在线发表

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