Genome-scale CRISPR screens identify host factors that promote human coronavirus infection
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Abstract
Background
The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design.
Methods
To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings.
Results
While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens.
Conclusions
Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.
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SciScore for 10.1101/2021.06.04.447090: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Virus stock generation and titer determination: SARS-CoV-2 strain UF-1 (GenBank accession number MT295464.1) was originally isolated from a COVID-19 patient at the University of Florida Health Shands Hospital via nasal swab (32) and manipulated in a Biosafety Level 3 (BSL3) laboratory at the Emerging Pathogens Institute under a protocol approved by the University of Florida Institutional Biosafety Committee.
Field Sample Permit: The sequencing was carried out at the Interdisciplinary Center for Biotechnology Research (ICBR; University of Florida) using a NovaSeq 6000 sequencer (Illumina).Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power … SciScore for 10.1101/2021.06.04.447090: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Virus stock generation and titer determination: SARS-CoV-2 strain UF-1 (GenBank accession number MT295464.1) was originally isolated from a COVID-19 patient at the University of Florida Health Shands Hospital via nasal swab (32) and manipulated in a Biosafety Level 3 (BSL3) laboratory at the Emerging Pathogens Institute under a protocol approved by the University of Florida Institutional Biosafety Committee.
Field Sample Permit: The sequencing was carried out at the Interdisciplinary Center for Biotechnology Research (ICBR; University of Florida) using a NovaSeq 6000 sequencer (Illumina).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
Antibodies Sentences Resources To confirm knockdown, cell lysates prepared from knockdown and control cells were tested by western blotting with antibodies directed to CTSL (Invitrogen, BMS1032) BMS1032suggested: NoneExperimental Models: Cell Lines Sentences Resources Virus stocks were prepared by infecting Vero E6 cells at MOI 0.01, centrifuging culture supernatants collected at 3 dpi for 5 mins at 1000 x g, and filtering through a 0.44 μm PVDF filter (Millipore) followed by a 0.22 μm PVDF filter (Restek). Vero E6suggested: NoneFor both libraries, lentiviruses were produced in HEK293T cells by co-transfection of library plasmids together with the packaging plasmid psPAX2 (Addgene 12260) and envelope plasmid pMD2. HEK293Tsuggested: NoneValidation of host factors in promoting viral infections: To validate selected host factors for their capacity to promote HCoV infection in vitro, we transduced HEK293T-hACE2 cells with lentivirus-packaged shRNAs targeting CTSL, CCZ1, or EDC4 (TRC Human shRNA Library collection) or the empty vector pLKO.1. HEK293T-hACE2suggested: RRID:CVCL_A7UK)Recombinant DNA Sentences Resources For both libraries, lentiviruses were produced in HEK293T cells by co-transfection of library plasmids together with the packaging plasmid psPAX2 (Addgene 12260) and envelope plasmid pMD2. psPAX2suggested: RRID:Addgene_12260)pMD2suggested: NoneValidation of host factors in promoting viral infections: To validate selected host factors for their capacity to promote HCoV infection in vitro, we transduced HEK293T-hACE2 cells with lentivirus-packaged shRNAs targeting CTSL, CCZ1, or EDC4 (TRC Human shRNA Library collection) or the empty vector pLKO.1. pLKO.1suggested: RRID:Addgene_13425)Software and Algorithms Sentences Resources The sgRNA sequences from the library were assembled into a Burrows-Wheeler index using the Bowtie build-index function and reads were aligned to the index. Bowtiesuggested: (Bowtie, RRID:SCR_005476)We submitted FastQ files to Gene expression omnibus (GSE: XXXXXX) and all CRISPR screen data to BioGRID ORCS database (https://orcs.thebiogrid.org/). BioGRIDsuggested: (BioGrid Australia, RRID:SCR_006334)Identification and testing inhibitors of host factors from CRISPR screens: Online databases and published literature were used to find small molecule inhibitors targeting a subset of top-scoring genes in the CRISPR screens. Onlinesuggested: (Globus Online, RRID:SCR_012284)These percentages were compared to the values obtained from virus infected-cell cytotoxicity values by one-way ANOVA using GraphPad Prism version 9. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)EC50 and IC50 values were calculated by transforming inhibitor concentrations to log then using the non-linear fit with variable slope function (GraphPad Prism version 9) to determine best fit variables using the percent of maximum SARS-CoV-2 induced cytotoxicity measurements at each drug concentration performed in technical duplicate. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)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.
Results from scite Reference Check: We found no unreliable references.
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