将 CRISPR/Cas 介导的末端解析与新型遗传学工作流程相结合,实现高多样性腺病毒文库

IF 4.6 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics
{"title":"将 CRISPR/Cas 介导的末端解析与新型遗传学工作流程相结合,实现高多样性腺病毒文库","authors":"Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics","doi":"10.1016/j.omtm.2024.101241","DOIUrl":null,"url":null,"abstract":"While recombinant Adenoviruses are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated terminal resolution aiding high-efficiency rescue of recombinant Adenoviruses from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based recombinant Adenoviruses libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement allows the introduction of >10ˆ6 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5x10ˆ4 unique recombinant Adenoviruses per cmˆ2 of transfected cell culture.","PeriodicalId":54333,"journal":{"name":"Molecular Therapy-Methods & Clinical Development","volume":null,"pages":null},"PeriodicalIF":4.6000,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries\",\"authors\":\"Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics\",\"doi\":\"10.1016/j.omtm.2024.101241\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"While recombinant Adenoviruses are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated terminal resolution aiding high-efficiency rescue of recombinant Adenoviruses from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based recombinant Adenoviruses libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement allows the introduction of >10ˆ6 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5x10ˆ4 unique recombinant Adenoviruses per cmˆ2 of transfected cell culture.\",\"PeriodicalId\":54333,\"journal\":{\"name\":\"Molecular Therapy-Methods & Clinical Development\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2024-03-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Therapy-Methods & Clinical Development\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1016/j.omtm.2024.101241\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Therapy-Methods & Clinical Development","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.omtm.2024.101241","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
引用次数: 0

摘要

虽然重组腺病毒被广泛应用于实验室和医学基因转移,但使用这种载体平台的基于文库的应用并不容易获得。最近,我们开发了一种新方法--CRISPR/Cas9 介导的末端解析,有助于从重组 DNA 中高效拯救重组腺病毒。在此,我们报告了一种基因工作流程,它允许利用高效的末端解析构建基于 BAC 的重组腺病毒文库。我们利用频繁出现的、预先存在的基因组序列来插入选择标记,将两个选定的目标位点补充到新型内切酶识别位点中。第二步,通过 Gibson 组装,用转基因或感兴趣的突变取代选择标记。我们的方法不会造成不必要的基因组脱靶突变,同时为基因修饰的位点和性质提供了极大的灵活性。我们将这种新的基因工作流程称为半位定向片段置换,它可以利用实验室规模的方法在 rAd 编码 BAC 中引入大于 10ˆ6 的独特修饰。为了证明 HFR 的威力,我们拯救了条形码病毒载体文库,在每厘米 2 的转染细胞培养物中产生了 2.5x10ˆ4 种独特的重组腺病毒。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries
While recombinant Adenoviruses are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated terminal resolution aiding high-efficiency rescue of recombinant Adenoviruses from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based recombinant Adenoviruses libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement allows the introduction of >10ˆ6 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5x10ˆ4 unique recombinant Adenoviruses per cmˆ2 of transfected cell culture.
求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Molecular Therapy-Methods & Clinical Development
Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
9.90
自引率
4.30%
发文量
163
审稿时长
12 weeks
期刊介绍: The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信