利用HDR增强模块化ssDNA供体提高同源定向修复效率-动脉网

Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor

Nature 等信源发布 2024-08-10 09:14

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AbstractDespite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors.

摘要尽管小分子和重组蛋白具有提高同源定向修复（HDR）效率的潜力，但目前设计和化学修饰的单链DNA（ssDNA）供体对于精确的基因编辑仍然不是最理想的。在这里，我们筛选了DNA修复相关蛋白的偏向ssDNA结合序列，并将RAD51首选序列工程化为ssDNA供体的HDR增强模块。

Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency.

具有这些模块的供体对RAD51表现出增强的亲和力，从而在与Cas9，nCas9和Cas12a合作时提高了各种基因组位点和细胞类型的HDR效率。通过与非同源末端连接抑制剂（NHEJ）或HDRobust策略相结合，这些模块化ssDNA供体的HDR效率高达90.03%（中位数74.81%）。

The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing..

针对内源性蛋白质的HDR增强模块可以实现无化学修饰的策略，以提高ssDNA供体进行精确基因编辑的功效。。

IntroductionPrecise gene editing is significant for both biological research and clinical gene therapy applications. Gene editing relies on intrinsic DNA repair pathways triggered by DNA damage, such as double-stranded break (DSB), site-specifically induced by programmable endonucleases1. Endogenous proteins are recruited to these sites, where they repair the DSB via two major pathways: error-prone non-homologous end joining (NHEJ) and precise homology-directed repair (HDR)2.

引言精确的基因编辑对于生物学研究和临床基因治疗应用都具有重要意义。基因编辑依赖于由DNA损伤触发的内在DNA修复途径，例如由可编程核酸内切酶特异性诱导的双链断裂（DSB）。内源性蛋白质被募集到这些位点，在那里它们通过两种主要途径修复DSB：易错的非同源末端连接（NHEJ）和精确的同源定向修复（HDR）2。

When these pathways function ineffectively, other error-prone repair pathways, microhomology-mediated end joining (MMEJ) and single-stranded annealing (SSA), evolved presumably to mediate repair1,3,4. NHEJ is initiated by the binding of the Ku70-Ku80 heterodimer to DSB ends and often induces insertions and deletions (indels).

当这些途径无效时，其他容易出错的修复途径，微同源介导的末端连接（MMEJ）和单链退火（SSA）可能会进化为介导修复1,3,4。NHEJ是由Ku70-Ku80异二聚体与DSB末端的结合引发的，通常会诱导插入和缺失（indels）。

In contrast, HDR utilizes proteins like CtIP, the MRN complex (MRE11-RAD50-NBS1), and RAD51 to repair the DSB precisely, using sister chromatids as templates2. Therefore, exogenously provided DNA donors containing the intended sequence can also be integrated at the target site via HDR, enabling precise gene editing5,6.

相反，HDR利用CtIP，MRN复合物（MRE11-RAD50-NBS1）和RAD51等蛋白质，以姐妹染色单体为模板，精确修复DSB 2。因此，含有预期序列的外源提供的DNA供体也可以通过HDR整合到靶位点，从而实现精确的基因编辑5,6。

Because the programmable endonucleases, Cas9 and Cas12a, have demonstrated high efficiency in inducing DSB, the relatively low efficiency of HDR compared to NHEJ has been a major bottleneck in achieving precise gene editing at desired loci. Recently, inhibition of error-prone repair pathways by dominant-negative 53BP1-fused Cas97, the small molecule M38148,9, and HDRobust strategy9 have been reported to shift the endogenous DNA repair pathway toward HDR, yielding high precise gene editing efficiency.

由于可编程核酸内切酶Cas9和Cas12a在诱导DSB方面表现出高效率，因此与NHEJ相比，HDR的效率相对较低，这是在所需基因座上实现精确基因编辑的主要瓶颈。最近，据报道，显性负性53BP1融合的Cas97，小分子M38148,9和HDRobust策略9抑制了易出错的修复途径，从而将内源性DNA修复途径转向HDR，从而产生了高精度的基因编辑效率。

Nonetheless, the involvement of exogenously delivered DNA donors remains a limiting step for optimal HDR efficiency5,6.Single-stranded DNA (ssDNA) donors generally .

尽管如此，外源递送的DNA供体的参与仍然是最佳HDR效率的限制步骤5,6。通常是单链DNA（ssDNA）供体。

Data availability

数据可用性

Next-generation sequencing data and mass spectrum data generated in this study have been deposited to the China National Center for Bioinformation under accession code PRJCA020579. All data generated or analysed during this study are included in this published article (and its supplementary information files). Source data are provided with this paper..

本研究中产生的下一代测序数据和质谱数据已保存在中国国家生物信息中心，登录号为PRJCA020579。本研究期间生成或分析的所有数据均包含在本文（及其补充信息文件）中。本文提供了源数据。。

Code availability

代码可用性

All code used for processing library data is available on GitHub55.

所有用于处理库数据的代码都可以在GitHub55上找到。

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Jin, Y. et al. Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor. HDR-boosting_modular_donors. 10.5281/zenodo.12635855, 2024.Download referencesAcknowledgementsWe thank all members of the De-Pei Liu laboratory for their helpful discussion about the project.

Jin，Y.等人。用HDR增强模块化ssDNA供体提高同源性定向修复效率。HDR-boosting\u modular\u捐赠者。10.5281/zenodo.126358552024。下载参考文献致谢我们感谢刘德培实验室的所有成员对该项目的有益讨论。

This work was supported by grants from the National Key Research and Development Project of China (grant no. 2021YFA0804903, to D.L.), the National Natural Science Foundation of China (grant no. 92149305, to D.L.), the Haihe Laboratory of Cell Ecosystem Innovation Fund (grant no. HH22KYZX0008, to D.L.), and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant no.

这项工作得到了国家重点研究发展项目（批准号2021YFA0804903，授予D.L.），国家自然科学基金（批准号92149305，授予D.L.），海河细胞生态系统创新基金实验室（批准号HH22KYZX0008，授予D.L.）和中国医学科学院医学科学创新基金（批准号：。

2021-I2M-1-016, 2022-I2M-2-001, and 2023-I2M-2-005, to D.L.).Author informationAuthor notesThese authors contributed equally: Ying-Ying Jin, Peng Zhang.These authors jointly supervised this work: De-Pei Liu, Hou-Zao Chen.Authors and AffiliationsState Key Laboratory of Common Mechanism Research for Major Diseases, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, ChinaYing-Ying Jin, Peng Zhang, Le-Le Liu, Xiang Zhao, Xiao-Qing Hu, Si-Zhe Liu, Ze-Kun Li, Qian Liu, Jian-Qiao Wang, De-Long Hao, Zhu-Qin Zhang, Hou-Zao Chen & De-Pei LiuMedical Epigenetics Research Center, Chinese Academy of Medical Sciences, Beijing, 100005, ChinaHou-Zao Chen & De-Pei LiuHaihe Laboratory of Cell Ecosystem, Tianjin, 300301, ChinaDe-Pei LiuAuthorsYing-Ying JinView author publicationsYou can also search for this author in.

2021-I2M-1-016、2022-I2M-2-001和2023-I2M-2-005，至D.L.）。作者信息作者注意到这些作者做出了同样的贡献：Ying Ying Jin，Peng Zhang。这些作者共同监督了这项工作：刘德培，陈厚早。作者及所属单位中国医学科学院北京协和医学院基础医学研究所生物化学与分子生物学系重大疾病共同机制研究国家重点实验室，北京，100005，中国盈盈金，张鹏，刘乐乐，赵翔，胡晓青，刘思哲，李泽坤，刘谦，王建桥，王德龙，张朱琴，陈厚早，刘德培中国医学科学院医学表观遗传学研究中心，北京，100005，陈厚早，刘德培细胞生态实验室，天津，300301，刘德培作者盈金观点作者出版物也可以在中搜索此作者。

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PubMed Google ScholarContributionsY.J. and P.Z. conceived the study and designed experiments; Y.J. performed the experimental studies; P.Z. conceptualized HDR-boosting modular ssDNA donor and analyzed the next-generation sequencing data; P.Z., L.L., X.Z., X.H., S.L., Z.L., Q.L., J.W., D.H., and Z.Z.

PubMed谷歌学术贡献。J、和P.Z.构思了这项研究并设计了实验；Y、 J.进行了实验研究；P、；P、 Z.，L.L.，X.Z.，X.H.，S.L.，Z.L.，Q.L.，J.W.，D.H.，和Z.Z。

assisted with experiments; Y.J. and P.Z. wrote the paper; D.L. and H.C. supervised the study.Corresponding authorsCorrespondence to.

协助实验；Y、 J.和P.Z.写了这篇论文；D、 L.和H.C.监督了这项研究。通讯作者通讯。

Hou-Zao Chen or De-Pei Liu.Ethics declarations

陈厚早或刘德培。道德宣言

Competing interests

相互竞争的利益

D.L., Y.J., P.Z., and H.C. have submitted two patent applications to the China National Intellectual Property Administration pertaining to the HDR-boosting modules and the combination of HDR-boosting modular ssDNA donor and M3814 inhibitor aspects of this work (application number 2023108476157 and 2024107260446).

D、 L.，Y.J.，P.Z。和H.C.已经向中国国家知识产权局提交了两项专利申请，涉及HDR增强模块以及HDR增强模块ssDNA供体和M3814抑制剂方面的组合（申请号2023108476157和2024107260446）。

The remaining authors declare no competing interests..

其余作者声明没有利益冲突。。

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Nature Communications thanks Xinyu Ling, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Nature Communications感谢凌新宇（Xinyu Ling）和另一位匿名审稿人对这项工作的同行评议做出的贡献。可以获得同行评议文件。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary informationSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1-6Reporting SummarySource dataSource DataRights and permissions.

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You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

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Reprints and permissionsAbout this articleCite this articleJin, YY., Zhang, P., Liu, LL. et al. Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor.

转载和许可本文引用本文Jin，YY。，张，P.，刘，LL。等。用HDR增强模块化ssDNA供体提高同源性定向修复效率。

Nat Commun 15, 6843 (2024). https://doi.org/10.1038/s41467-024-50788-xDownload citationReceived: 02 October 2023Accepted: 22 July 2024Published: 10 August 2024DOI: https://doi.org/10.1038/s41467-024-50788-xShare 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》156843（2024）。https://doi.org/10.1038/s41467-024-50788-xDownload引文接收日期：2023年10月2日接收日期：2024年7月22日发布日期：2024年8月10日OI：https://doi.org/10.1038/s41467-024-50788-xShare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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