Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.
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SciScore for 10.1101/2020.09.23.309849: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Transfection: The CPER products (25 μl out of a 50 μl reaction volume) were transfected into HEK293-3P6C33 cells or BHK-21 cells with Trans IT LT-1 (Mirus), following the manufacturer’s protocols. BHK-21suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)At 6 hours post-transfection, the culture supernatants of HEK293-3P6C33 cells were replaced with DMEM containing 2% FBS and doxycycline hydrochloride (1 μg/ml), and BHK-21 cells were … SciScore for 10.1101/2020.09.23.309849: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Transfection: The CPER products (25 μl out of a 50 μl reaction volume) were transfected into HEK293-3P6C33 cells or BHK-21 cells with Trans IT LT-1 (Mirus), following the manufacturer’s protocols. BHK-21suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)At 6 hours post-transfection, the culture supernatants of HEK293-3P6C33 cells were replaced with DMEM containing 2% FBS and doxycycline hydrochloride (1 μg/ml), and BHK-21 cells were overlaid by VeroE6/TMPRSS2 cells. HEK293-3P6C33suggested: NoneThe culture supernatants of cells were inoculated onto VeroE6/TMPRSS2 cells in 96-well plates after ten-fold serial dilution with DMEM containing 2% FBS, and the infectious titers were determined at 72 hours post-infection (hpi). VeroE6/TMPRSS2suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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