A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing
A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing
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DOI：
10.1038/s41586-020-2477-4
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发表时间：
2020-07-08
期刊：
影响因子：
64.8
通讯作者：
Liu, David R.
中科院分区：
文献类型：
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作者：
Mok, Beverly Y.;de Moraes, Marcos H.;Liu, David R.
细菌毒素代表了一个巨大的生物化学多样性库，可以重新用于生物医学应用。这些蛋白质包括脱氨酶超家族的一组预测的细菌间毒素，其成员已在基因编辑技术中找到应用（1，2）。由于先前描述的胞苷脱氨酶作用于单链核酸（3），因此它们在碱基编辑中的使用需要双链DNA（dsDNA）的解旋-例如通过CRISPR-Cas9系统。然而，线粒体DNA（mtDNA）内的碱基编辑迄今为止受到与将引导RNA递送到线粒体中相关的挑战的阻碍（4）。因此，迄今为止，对mtDNA的操作仅限于通过设计核酸酶靶向破坏线粒体基因组（9，10）。在此，我们描述了一种细菌间毒素，我们将其命名为DddA，其催化dsDNA内胞苷的脱氨基作用。我们设计了无毒且无活性的分裂-DddA半体，直到通过相邻结合的可编程DNA结合蛋白在靶DNA上聚集在一起。分裂DddA半，转录激活因子样效应阵列蛋白和尿嘧啶糖基化酶抑制剂的融合导致无RNA的DddA衍生的胞嘧啶碱基编辑器（DdCBE），其催化人mtDNA中C中心点G到T中心点A的转换，具有高靶特异性和产物纯度。我们使用DdCBE来模拟人类细胞中与疾病相关的mtDNA突变，导致呼吸速率和氧化磷酸化的变化。无CRISPR的DdCBE能够精确操纵mtDNA，而不是消除由靶向核酸酶切割产生的mtDNA拷贝，这对线粒体疾病的研究和潜在治疗具有广泛的意义。
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 techniques(1,2). Because previously described cytidine deaminases operate on single-stranded nucleic acids(3), 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 mitochondria(4). As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases(9,10).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 C center dot G-to-T center dot A 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.