Engineered IscB–ωRNA system with expanded target range for base editing

IF 12.9 1区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Nature chemical biology Pub Date : 2024-08-15 DOI:10.1038/s41589-024-01706-1

Qingquan Xiao, Guoling Li, Dingyi Han, Haoqiang Wang, Mingyu Yao, Tingting Ma, Jingxing Zhou, Yu Zhang, Xiumei Zhang, Bingbing He, Yuan Yuan, Linyu Shi, Tong Li, Hui Yang, Jinhai Huang, Hainan Zhang

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Abstract

As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.

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工程化 IscB-ωRNA 系统扩大了碱基编辑的目标范围

作为 Cas9 核酸酶的进化祖先，IscB 蛋白是一种紧凑型 RNA 引导的 DNA 内切酶和缺口酶，因此是碱基编辑的有力候选者。然而，狭窄的靶向范围限制了 IscB 系统的应用；因此，有必要找到更多可识别不同靶邻接基序（TAM）的 IscB。在这里，我们从未培殖微生物中鉴定出了19种未表征的IscB蛋白中的10种，它们在哺乳动物细胞中具有活性。通过蛋白质和ωRNA工程，我们进一步增强了IscB直向同源物IscB.m16的活性，并将其TAM范围从MRNRAA扩大到NNNGNA，从而产生了一种名为IscB.m16*的变体。通过将脱氨酶结构域与 IscB.m16* 缺口酶融合，我们产生了 IscB.m16* 衍生的碱基编辑器，它们在哺乳动物细胞中表现出强大的碱基编辑效率，并通过单次腺相关病毒递送有效恢复了患病小鼠的杜氏肌营养不良症蛋白。因此，这项研究为基础研究和治疗应用建立了一套紧凑的碱基编辑工具。

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来源期刊

Nature chemical biology 生物-生化与分子生物学

CiteScore

23.90

自引率

1.40%

发文量

238

审稿时长

12 months

期刊介绍： Nature Chemical Biology stands as an esteemed international monthly journal, offering a prominent platform for the chemical biology community to showcase top-tier original research and commentary. Operating at the crossroads of chemistry, biology, and related disciplines, chemical biology utilizes scientific ideas and approaches to comprehend and manipulate biological systems with molecular precision. The journal embraces contributions from the growing community of chemical biologists, encompassing insights from chemists applying principles and tools to biological inquiries and biologists striving to comprehend and control molecular-level biological processes. We prioritize studies unveiling significant conceptual or practical advancements in areas where chemistry and biology intersect, emphasizing basic research, especially those reporting novel chemical or biological tools and offering profound molecular-level insights into underlying biological mechanisms. Nature Chemical Biology also welcomes manuscripts describing applied molecular studies at the chemistry-biology interface due to the broad utility of chemical biology approaches in manipulating or engineering biological systems. Irrespective of scientific focus, we actively seek submissions that creatively blend chemistry and biology, particularly those providing substantial conceptual or methodological breakthroughs with the potential to open innovative research avenues. The journal maintains a robust and impartial review process, emphasizing thorough chemical and biological characterization.

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