Julian Fischer, Ariana Fedotova, Lena Jaki, Erwan Sallard, Anja Erhardt, Jonas Fuchs, Zsolt Ruzsics
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引用次数: 0
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.
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.