A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

Rihn, Suzannah J

; Merits, Andres; Bakshi, Siddharth; Turnbull, Matthew L

; Wickenhagen, Arthur

; Alexander, Akira JT

; Baillie, Carla; Brennan, Benjamin

; Brown, Fiona; Brunker, Kirstyn; +72 more...Bryden, Steven R

; Burness, Kerry A; Carmichael, Stephen; Cole, Sarah J; Cowton, Vanessa M; Davies, Paul

; Davis, Chris; De Lorenzo, Giuditta

; Donald, Claire L

; Dorward, Mark; Dunlop, James I; Elliott, Matthew; Fares, Mazigh; da Silva Filipe, Ana

; Freitas, Joseph R; Furnon, Wilhelm

; Gestuveo, Rommel J

; Geyer, Anna; Giesel, Daniel; Goldfarb, Daniel M

; Goodman, Nicola

; Gunson, Rory; Hastie, C James

; Herder, Vanessa

; Hughes, Joseph

; Johnson, Clare; Johnson, Natasha

; Kohl, Alain; Kerr, Karen; Leech, Hannah

; Lello, Laura Sandra; Li, Kathy; Lieber, Gauthier

; Liu, Xiang; Lingala, Rajendra; Loney, Colin; Mair, Daniel

; McElwee, Marion J; McFarlane, Steven; Nichols, Jenna

; Nomikou, Kyriaki

; Orr, Anne; Orton, Richard J

; Palmarini, Massimo; Parr, Yasmin A

; Pinto, Rute Maria

; Raggett, Samantha; Reid, Elaine; Robertson, David L

; Royle, Jamie

; Cameron-Ruiz, Natalia

; Shepherd, James G

; Smollett, Katherine

; Stewart, Douglas G; Stewart, Meredith

; Sugrue, Elena

; Szemiel, Agnieszka M; Taggart, Aislynn

; Thomson, Emma C

; Tong, Lily

; Torrie, Leah S; Toth, Rachel; Varjak, Margus; Wang, Sainan; Wilkinson, Stuart G

; Wyatt, Paul G; Zusinaite, Eva; Alessi, Dario R; Patel, Arvind H

; Zaid, Ali

; Wilson, Sam J

; and Mahalingam, Suresh

(2021) A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS biology, 19 (2). e3001091-. ISSN 1544-9173 DOI: 10.1371/journal.pbio.3001091

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The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.