Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics
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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。
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.
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.