设计TadA直系同源胞嘧啶碱基编辑器，无基序偏好和腺苷活性限制-动脉网

Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation

Nature 等信源发布 2024-09-16 15:54

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AbstractThe engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope.

摘要胞嘧啶碱基编辑器（CBE）中使用的工程化TadA变体具有独特的优势，与依赖天然脱氨酶的胞嘧啶碱基编辑器相比，其尺寸更小，脱靶效应更少。然而，目前的TadA变体表现出对具有特定基序序列的DNA中碱基编辑的偏好，并且具有双重脱氨酶活性，作用于相邻位置的胞嘧啶和腺苷，限制了它们的应用范围。

To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression.

。值得注意的是，将aTdCBE递送至杜兴氏肌营养不良症（DMD）小鼠的人源化小鼠模型可实现强大的外显子55跳跃和肌营养不良蛋白表达的恢复。

Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications..

我们在设计用于胞嘧啶编辑的TadA直系同源物方面的进步丰富了基因编辑疗法和其他潜在应用的基础编辑工具包。。

IntroductionDNA base editors composed of Cas9 nickase and deaminases can perform specific single base conversion on target sequences without requiring DNA double-stranded breaks1. The main effector domains of the cytosine base editor (CBE) and adenine base editor (ABE) are cytidine and adenine deaminase, catalyzing the base conversion from C·G to T·A and A·T to G·C, respectively.

引言由Cas9切口酶和脱氨酶组成的DNA碱基编辑器可以在不需要DNA双链断裂的情况下对靶序列进行特定的单碱基转化1。胞嘧啶碱基编辑器（CBE）和腺嘌呤碱基编辑器（ABE）的主要效应域是胞苷和腺嘌呤脱氨酶，分别催化从C·G到T·A和A·T到G·C的碱基转化。

Cytidine deaminases derived from proteins such as APOBEC, AID, and CDA naturally possess cytosine deamination activity1,2,3. The adenosine deaminases TadA of ABEs were engineered from tRNA-specific adenosine deaminase for adenosine base editing of DNA4,5. After removing uracil glycosylase inhibitor (UGI) from CBEs, C-to-G base editors (CGBEs) were developed to catalyze the conversion of bases from C.G to T.A6,7.By introducing stop codons (CAA/CAG/CGA to TAA/TAG/TGA), CBEs have shown great potential in the treatment of common diseases such as T-cell acute lymphoblastic leukemia8, Hepatitis B9,10, and acquired immunodeficiency syndrome11.

源自APOBEC，AID和CDA等蛋白质的胞苷脱氨酶天然具有胞嘧啶脱氨活性1,2,3。。从CBE中去除尿嘧啶糖基化酶抑制剂（UGI）后，开发了C-to-G碱基编辑器（CGBE），以催化碱基从C.G到T.A6的转化，7。通过引入终止密码子（CAA/CAG/CGA到TAA/TAG/TGA），CBE在治疗常见疾病如T细胞急性淋巴细胞白血病8，乙型肝炎9,10和获得性免疫缺陷综合征11方面显示出巨大潜力。

Although high on-target DNA editing efficiency can be achieved with traditional CBEs12, they can also exhibit different types of DNA and RNA off-target effects13,14,15,16. Compared to natural cytidine deaminases with high intrinsic single-stranded DNA (ssDNA) affinity, TadA of ABE exhibited undetectable guide-independent off-target effects at DNA and RNA levels17.

虽然传统的CBEs12可以实现高的靶向DNA编辑效率，但它们也可以表现出不同类型的DNA和RNA脱靶效应13,14,15,16。与具有高内在单链DNA（ssDNA）亲和力的天然胞苷脱氨酶相比，ABE的TadA在DNA和RNA水平上表现出不可检测的指导独立的脱靶效应17。

Previous studies reported that the TadA8e variant derived from E. coli TadA exhibited partial cytidine deaminase activity for TC sequences within the editing window18. Recently, various CBEs, including Td-CBEmax and TadCBEd, have been developed by engineering TadA8e into a cytosine deaminase, demonstrating the engineering plasticity of TadA for versatile base editors development19,20,21.

先前的研究报道，源自大肠杆菌TadA的TadA8e变体在编辑窗口内对TC序列表现出部分胞苷脱氨酶活性18。最近，通过将TadA8e工程化为胞嘧啶脱氨酶，已经开发了各种CBE，包括Td-CBEmax和TadCBEd，证明了TadA对多功能基础编辑器开发的工程可塑性19,20,21。

In addition, T.

此外，T。

Data availability

数据可用性

The deep-seq data and RNA-seq data generated in this study have been deposited in the NCBI database under accession code PRJNA1061823. Source data are provided with this paper.

本研究中产生的deep-seq数据和RNA-seq数据已保存在NCBI数据库中，登录号为PRJNA1061823。本文提供了源数据。

Code availability

代码可用性

The scripts and codes used in this manuscript are available at zenodo.org (accession code: 13337534).

本手稿中使用的脚本和代码可在zenodo.org上获得（登录号：13337534）。

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Download referencesAcknowledgementsThis work was supported by HUIDAGENE Therapeutics Inc, Project of National Natural Science Foundation of China (82271048 to J.H., 82301215 to G.Z.), Science and Technology Commission of Shanghai Municipality (22S11900200 and 23XD1420500 to J.H.), Seed Industry Vitalization Program of Jiangsu Province (JBGS[2021]025 to E.Z.), Jiangsu Provincial Key Research and Development Program (BE2021372 to E.Z.) and the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-SCAB-202301 to T.Y.).Author informationAuthor notesThese authors contributed equally: Guoling Li, Xue Dong, Jiamin Luo, Tanglong Yuan, Tong Li, Guoli Zhao.Authors and AffiliationsHuidaGene Therapeutics Co., Ltd., Shanghai, 200131, ChinaGuoling Li, Xue Dong, Jiamin Luo, Tong Li, Hainan Zhang, Jingxing Zhou, Shuna Cui, Haoqiang Wang, Yin Wang, Yuyang Yu, Yuan Yuan & Yingsi ZhouShenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, ChinaTanglong Yuan & Erwei ZuoEye Institute and Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Key Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, 200030, ChinaGuoli Zhao, Zhenhai Zeng & Jinhai HuangShanghai Research Center of Ophthalmology and Optometry, Shanghai, 200030, ChinaGuoli Zhao, Zhenhai Zeng & Jinhai HuangLingang Laboratory, Shanghai, ChinaChunlong XuAuthorsGuoling LiView author publicationsYou can also search for this author in.

下载参考文献致谢这项工作得到了惠达基因治疗有限公司，国家自然科学基金项目（82271048至J.H.，82301215至G.Z.），上海市科学技术委员会（22S11900200和23XD1420500至J.H.），江苏省种业振兴计划（JBGS[2021]025至E.Z.），江苏省重点研究与发展计划（BE2021372至E.Z.）和中国农业科学院创新计划（CAAS-SCAB-202301至T.Y.）的支持。作者信息作者注意到这些作者做出了同样的贡献：李国玲，薛东，罗家民，袁唐龙，李彤，赵国丽。作者和所属单位上海水大基因治疗有限公司，200131，中国李国玲，薛东，罗家民，童丽，海南张，周景兴，崔淑娜，王浩强，尹旺，余阳宇，袁元和周英思深圳分院，广东岭南现代农业实验室，农业和农村事务部合成生物学重点实验室，中国农业科学院深圳农业基因组研究所，深圳，中国唐龙园和二卫左业研究所，复旦大学眼科和耳鼻喉科医院眼科，中国医学科学院近视重点实验室，上海，200030，赵国力，曾振海＆JinhaiHuangshanghai眼科和验光研究中心，上海，200030，中国赵国力，曾振海＆JinhaiHuanglingang实验室，上海，中国徐春龙作者郭玲LiView作者出版物您也可以在中搜索该作者。

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PubMed Google ScholarContributionsY.Z. conceived the project. G.L., X.D., J.L., J.H. and G.Z. designed experiments. X.D., J.L. and G.L. performed protein engineering and endogenous sites base editing assay. T.L. and G.Z. performed the off-target assay. Y.Z. performed bioinformatics analysis.

PubMed谷歌学术贡献。Z、构思了这个项目。G、 L.，X.D.，J.L.，J.H.和G.Z.设计了实验。十、 D.，J.L.和G.L.进行了蛋白质工程和内源性位点碱基编辑测定。T、 L.和G.Z.进行了脱靶测定。Y、 Z.进行了生物信息学分析。

E.Z. and T.Y. performed the high-throughput library experiments. G.L., Y.Yu., and Y.W. performed in vivo virus injection, tissue dissection, histological immunostaining and muscle function experiments. H.Z., J.Z., Z.Z., H.W., S.C., and Y.Yu. assisted with experiments. Y.Z., J.H., and C.X. supervised the whole project.

E、 Z.和T.Y.进行了高通量文库实验。G、 L.，Y.Yu。，和Y.W.进行了体内病毒注射，组织解剖，组织学免疫染色和肌肉功能实验。H、 Z.，J.Z.，Z.Z.，H.W.，S.C。和Y.Yu。协助实验。Y、 Z.，J.H。和C.X.监督了整个项目。

Y.Z., C.X., G.L. and Y.Yuan. wrote the manuscript with data contributed by all authors who participated in the project.Corresponding authorsCorrespondence to.

Y、 Z.，C.X.，G.L.和Y.Yuan。用参与该项目的所有作者提供的数据撰写了手稿。通讯作者通讯。

Erwei Zuo, Chunlong Xu, Jinhai Huang or Yingsi Zhou.Ethics declarations

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X.D, G.L. and Y.Z. have submitted a patent application to the China National Intellectual Property Administration pertaining to the deaminase engineering aspect of this work (application number PCT/CN2024/078613). The remaining authors declare no competing interests.

十、 D，G.L.和Y.Z.已经向中国国家知识产权局提交了一份关于这项工作的脱氨酶工程方面的专利申请（申请号PCT/CN2024/078613）。其余作者声明没有利益冲突。

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Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

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Reprints and permissionsAbout this articleCite this articleLi, G., Dong, X., Luo, J. et al. Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation.

转载和许可本文引用本文Li，G.，Dong，X.，Luo，J。等人。工程TadA直系同源物衍生的胞嘧啶碱基编辑器，无基序偏好和腺苷活性限制。

Nat Commun 15, 8090 (2024). https://doi.org/10.1038/s41467-024-52485-1Download citationReceived: 08 February 2024Accepted: 10 September 2024Published: 16 September 2024DOI: https://doi.org/10.1038/s41467-024-52485-1Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

《国家公社》158090（2024）。https://doi.org/10.1038/s41467-024-52485-1Download引文接收日期：2024年2月8日接受日期：2024年9月10日发布日期：2024年9月16日OI：https://doi.org/10.1038/s41467-024-52485-1Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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