Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor

  1. Oded Danziger
  2. Roosheel S. Patel
  3. Emma J. DeGrace
  4. Mikaela R. Rosen
  5. Brad R. Rosenberg

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Abstract

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

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  1. Version published to 10.1371/journal.ppat.1010464
  2. ScreenIT

    SciScore for 10.1101/2021.09.22.461286: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: A549 and derived cell lines (ACE2-expressing cell lines with or without CRISPRa expression and ΔSTAT150 counterparts) were validated by short tandem repeat (STR) analysis (all confirmed 100% match to A549, CVCL_0023).
    Contamination: All cell lines were routinely tested (Boca Scientific #50-168-5641) negative for Mycoplasma contamination.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    This transfection mix was added to 1.5×106 Lenti-X 293T cells (Takara Bio #632180, plated in 6 well plates 18 hours prior to transfection) in1 ml of 10% FBS DMEM for 8 hours after which transfection media was removed and replaced with 2ml of 10% FBS DMEM.
    293T
    suggested: None
    Generation of A549-SunTag and A549ΔSTAT1-SunTag cells: To generate A549-SunTag and A549ΔSTAT1-SunTag cells, we transduced A549 or A549ΔSTAT1 cells with lentiviruses encoding the Dox-inducible transactivator component of SunTag (pCW-TRE-scFv-SunTag).
    A549ΔSTAT1-SunTag
    suggested: None
    A549ΔSTAT1
    suggested: None
    Curation and Cloning of the ISG library: To assemble the list of ISGs targeted by the gRNA library, an established list of ISGs3,4 was combined with a list of genes upregulated (RNA-Seq log2 fold-change >2, adjusted p value < 0.05) after 6 or 48 hours of IFN stimulation in A549 cells50.
    A549
    suggested: None
    At 72 hours post-infection, Vero-E6 cells were fixed in 4% Paraformaldehyde at room temperature for 24 hours, washed twice with PBS and stained with 1% Crystal violet (Sigma # C0775) for 15 minutes.
    Vero-E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Generation of lentiviruses and viral transduction: To generate lentiviruses for gRNA or cDNA expression, a mix of 2.5μg of the desired transfer vector, 2μg psPAX2 and 0.8μg of pMD2.
    psPAX2
    suggested: RRID:Addgene_12260)
    pMD2
    suggested: None
    Cloning of inducible CRISPRa-SunTag system components: To generate the plasmids for the inducible CRISPRa system, we re-engineered an existing CRISPRa technology45 to enable Dox-inducible expression and independent component construct antibiotic selection. pCW-TRE, a Dox-inducible expression vector, was generated by modifying pCW-Cas9-Blast (a gift from Mohan Babu, Addgene # 83481) to include a single BamHi site.
    pCW-Cas9-Blast
    suggested: RRID:Addgene_83481)
    Next, the antibody component of the SunTag system (pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS45, a gift from Ron Vale, Addgene #60904) was digested with EcoRI+NotI and subcloned into the blunted BamHi site of pCW-TRE.
    pCW-TRE
    suggested: None
    The nuclease-inactive Cas9 fused to the SunTag scaffold (dCas9-SunTag derived from pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP45, a gift from Ron Vale, Addgene #60903) was assembled in-frame with a hygromycin resistance gene into pHR-PGK (a gift from Wendell Lim, Addgene #7912093), generating pHR-PGK-dCas9-SunTag-P2A-HygR.
    pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP45
    suggested: None
    pHR-PGK-dCas9-SunTag-P2A-HygR
    suggested: None
    Generation of A549-SunTag and A549ΔSTAT1-SunTag cells: To generate A549-SunTag and A549ΔSTAT1-SunTag cells, we transduced A549 or A549ΔSTAT1 cells with lentiviruses encoding the Dox-inducible transactivator component of SunTag (pCW-TRE-scFv-SunTag).
    pCW-TRE-scFv-SunTag
    suggested: None
    Generation of ACE2 expressing cell lines: To generate ACE2-expressing A549 cell lines, the human ACE2 coding sequence (RefSeq accession NM_001371415.1) was PCR amplified and cloned into the BamHi site of lentiviral vector pHR-PGK (Addgene #79120).
    pHR-PGK
    suggested: None
    Cloning procedures for individual guide RNAs: Cloning of individual gRNAs into CROP-seq-opti vectors was performed as previously described96,97.
    CROP-seq-opti
    suggested: RRID:Addgene_106280)
    Genetic sequences were synthesized/amplified with homology overhangs complementary to the overhangs of EcoRI+BamHi digested pLVX-TetOne-Puro (Takara # 631849).
    pLVX-TetOne-Puro
    suggested: RRID:Addgene_124797)
    Software and Algorithms
    SentencesResources
    Generation of ACE2 expressing cell lines: To generate ACE2-expressing A549 cell lines, the human ACE2 coding sequence (RefSeq accession NM_001371415.1) was PCR amplified and cloned into the BamHi site of lentiviral vector pHR-PGK (Addgene #79120).
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    Inducible CRISPRa ISG screen data processing and analysis: Illumina BCL sequence files were converted to FASTQ format with the bcl2fastq tool (v2.20.0.422, Illumina).
    bcl2fastq
    suggested: (bcl2fastq , RRID:SCR_015058)
    For all viral infections, analysis was performed with FlowJo software (Version 10.7.1, Becton Dickinson), excluding cell doublets and debris and gating according to mock infected populations (Supplementary Fig. 4).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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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  3. Version published to 10.1101/2021.09.22.461286v1 on bioRxiv