دورية أكاديمية
Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform
| العنوان: | Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform |
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| المؤلفون: | Enja Tatjana Kipfer, David Hauser, Martin J Lett, Fabian Otte, Lorena Urda, Yuepeng Zhang, Christopher MR Lang, Mohamed Chami, Christian Mittelholzer, Thomas Klimkait |
| المصدر: | eLife, Vol 12 (2023) |
| بيانات النشر: | eLife Sciences Publications Ltd, 2023. |
| سنة النشر: | 2023 |
| المجموعة: | LCC:Medicine LCC:Science LCC:Biology (General) |
| مصطلحات موضوعية: | SARS-CoV-2, reverse genetics, RNA virus, Chikungunya, Dengue, One Health, Medicine, Science, Biology (General), QH301-705.5 |
| الوصف: | Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the coronavirus disease 2019 pandemic, already established methods were challenged by the large genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment – harboring all heterologous sequences – viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 2050-084X |
| Relation: | https://elifesciences.org/articles/89035; https://doaj.org/toc/2050-084X |
| DOI: | 10.7554/eLife.89035 |
| URL الوصول: | https://doaj.org/article/54577bf2a8de47538fbd50df01e9ec39 |
| رقم الأكسشن: | edsdoj.54577bf2a8de47538fbd50df01e9ec39 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 2050084X |
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| DOI: | 10.7554/eLife.89035 |