A scalable pipeline for SARS-CoV-2 replicon construction based on de-novo synthesis
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Abstract
Replicons are synthetic viral RNA molecules that recapitulate the self-replicating activities of the virus but are missing its infectivity potential. Here, we report on a scalable pipeline to generate a replicon of any SARS-CoV-2 strain using de-novo synthesis. Our pipeline relies only on publicly available sequencing data without requiring access to any material, simplifying logistical and bureaucratic issues of sample acquisition. In addition, our system retains the nucleotide sequence of most of the SARS-CoV-2 full genome and therefore better captures its underlying genomic and biological functions as compared to the popular pseudotypes or any replicon system published to-date. We utilized our system to synthesize a SARS-CoV-2 non-infectious version of the Beta strain. We then confirmed that the resulting RNA molecules are non-infectious and safe to handle in a BSL2/CL2 facility. Finally, we show that our replicon can be specifically inhibited by molnupiravir and RNAi treatments, demonstrating its utility for drug research and development.
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SciScore for 10.1101/2022.02.05.478644: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources 12 hours later, the media was replaced to remove dead cells arising from electroporation. siRNA testing: 0.5 million HEK293T cells, 0.2ug replicon and 100nM siRNA duplex were mixed in the electroporation buffer (Neon Transfection Kit, Thermofisher). HEK293Tsuggested: NoneRecombinant DNA Sentences Resources Cloning was carried out by adding the two 15kb fragments (80fmol each) to 40fmol pSMART BAC vector (Lucigen) and incubating them with a proprietary assembly mix … SciScore for 10.1101/2022.02.05.478644: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources 12 hours later, the media was replaced to remove dead cells arising from electroporation. siRNA testing: 0.5 million HEK293T cells, 0.2ug replicon and 100nM siRNA duplex were mixed in the electroporation buffer (Neon Transfection Kit, Thermofisher). HEK293Tsuggested: NoneRecombinant DNA Sentences Resources Cloning was carried out by adding the two 15kb fragments (80fmol each) to 40fmol pSMART BAC vector (Lucigen) and incubating them with a proprietary assembly mix (Twist) at 65°C for 30 minutes. pSMART BACsuggested: NoneBriefly, 4ug plasmid was linearised with 10ul of AgeI at 37°C for 4 hours. 4ugsuggested: NoneSoftware and Algorithms Sentences Resources We constructed a multiple sequence alignment using the “-diags” option in MUSCLE v3.8.1551 18. MUSCLEsuggested: (MUSCLE, RRID:SCR_011812)Second, we obtained the reference genome sequence of each SARS-CoV-2 gene (ORF1ab, S, N, E, ORF3a, ORF8, M, ORF7a, ORF6, ORF7b, ORF10) from the reference genome sequence, based on gene coordinates obtained from the NCBI gene database (https://www.ncbi.nlm.nih.gov/gene/?term=NC_045512.2). NCBIsuggested: (NCBI, RRID:SCR_006472)Plasmids were NGS library prepped with Nextera XT DNA Sample Prep Kit (Illumina) and sequenced 2x×150bp by MiSeq (Illumina). NGSsuggested: (ANGSD, RRID:SCR_021865)MiSeqsuggested: (A5-miseq, RRID:SCR_012148)NGS analysis was done by alignment with BWA-MEM to the reference and the bam file was visualized on Integrative Genomics Viewer (IGV) BWA-MEMsuggested: (Sniffles, RRID:SCR_017619)Transfection: The cells were harvested using TrypLE Express (ThermoFisher), washed with 1X PBS and resuspended in 10ul of electroporation buffer (Neon Transfection Kit, Thermofisher). ThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Replicon coverage pipeline: We trimmed the adapters of pair-end reads with minimal length of 5 bases using cutadapt v3.519 and mapped the remaining reads to the replicon reference using Bowtie2 v2.4.420. Bowtie2suggested: (Bowtie 2, RRID:SCR_016368)Next, we converted the SAM output files to sorted BAM files using SAMtools v1.1121 and extracted the coverage as Wig files using the IGVTools22 count function, with a window size of 1 for reads with a minimal mapping quality of 1. SAMsuggested: (SAM, RRID:SCR_010951)SAMtoolssuggested: (SAMTOOLS, RRID:SCR_002105)Since the siRNA degraded a large proportion of the replicon genome, we calibrated the null distribution of DESeq2 using the control genes parameter, using the first 200 tiles (corresponding to the first 20,000 bases of the replicon genome). DESeq2suggested: (DESeq, RRID:SCR_000154)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.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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