Efficient Reverse Genetic Systems for Rapid Genetic Manipulation of Emergent and Preemergent Infectious Coronaviruses

Methods in Molecular Biology
Adam S CockrellRalph S. Baric

Abstract

Emergent and preemergent coronaviruses (CoVs) pose a global threat that requires immediate intervention. Rapid intervention necessitates the capacity to generate, grow, and genetically manipulate infectious CoVs in order to rapidly evaluate pathogenic mechanisms, host and tissue permissibility, and candidate antiviral therapeutic efficacy. CoVs encode the largest viral RNA genomes at about 28-32,000 nucleotides in length, and thereby complicate efficient engineering of the genome. Deconstructing the genome into manageable fragments affords the plasticity necessary to rapidly introduce targeted genetic changes in parallel and assort mutated fragments while maximizing genome stability over time. In this protocol we describe a well-developed reverse genetic platform strategy for CoVs that is comprised of partitioning the viral genome into 5-7 independent DNA fragments (depending on the CoV genome), each subcloned into a plasmid for increased stability and ease of genetic manipulation and amplification. Coronavirus genomes are conveniently partitioned by introducing type IIS or IIG restriction enzyme recognition sites that confer directional cloning. Since each restriction site leaves a unique overhang between adjoining fragments, ...Continue Reading

Citations

Mar 9, 2019·Expert Opinion on Drug Discovery·Allison L Totura, Sina Bavari
Jul 2, 2020·International Journal of Molecular Sciences·Alejandro LlanesRicardo Lleonart
Jul 26, 2018·Mammalian Genome : Official Journal of the International Mammalian Genome Society·Adam S CockrellRalph S Baric
Mar 18, 2021·Brazilian Journal of Medical and Biological Research = Revista Brasileira De Pesquisas Médicas E Biológicas·J E Belizário
Jun 10, 2021·Nature Communications·Alberto A AmarillaAlexander A Khromykh

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Methods Mentioned

BETA
transfection
in
in vitro transcription
PCR
electrophoresis
restriction

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