key: cord-0278868-9rvrcyh0
authors: Danziger, Oded; Patel, Roosheel S; DeGrace, Emma J; Rosen, Mikaela R; Rosenberg, Brad R
title: Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor
date: 2021-09-23
journal: bioRxiv
DOI: 10.1101/2021.09.22.461286
sha: ee634176bdce7e5adeb92883fb6a7427fb48ffeb
doc_id: 278868
cord_uid: 9rvrcyh0

Interferons establish an antiviral state in responding cells through the induction of hundreds of interferon-stimulated genes (ISGs). ISGs antagonize viral pathogens directly through diverse mechanisms acting at different stages of viral life cycles, and indirectly by modulating cell cycle and promoting programmed cell death. The mechanisms of action and viral specificities for most ISGs remain incompletely understood. To enable the high throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating the potentially confounding effects of endogenous IFN and potential antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG induction in isogenic cell lines with and without the capacity to respond to IFN. Engineered CRISPRa cell lines demonstrated inducible, robust, and specific gRNA-directed expression of ISGs, which are functional in restricting viral infection. Using this platform, we screened for ISGs that restrict SARS-CoV-2, the causative agent of the COVID-19 pandemic. Results included ISGs previously described to restrict SARS-CoV-2 as well as multiple novel candidate antiviral factors. We validated a subset of candidate hits by complementary targeted CRISPRa and ectopic cDNA expression infection experiments, which, among other hits, confirmed OAS1 as a SARS-CoV-2 restriction factor. OAS1 exhibited strong antiviral effects against SARS-CoV-2, and these effects required OAS1 catalytic activity. These studies demonstrate a robust, high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by the identification of multiple novel SARS-CoV-2 restriction factors.

Interferons (IFN) act as key mediators of the host response to viral pathogens by establishing a general antiviral state through the coordinated induction of hundreds of interferon stimulated genes (ISGs) in infected and uninfected "bystander" cells 1 [3] [4] [5] [6] [7] . Other individual ISGs have been found to restrict or even to enhance the replication of specific viruses. In addition to direct antiviral effectors, the ISG repertoire also includes genes that induce antiproliferative and/or proapoptotic programs in response to viral infection or DNA damage, thereby limiting viral spread and impeding oncogenesis [8] [9] [10] [11] .

A robust IFN response is critical for host defense against novel respiratory viruses to which immune memory from prior exposure has not been established 12 20 . Additionally, several studies have identified autoantibodies against IFN as a significant negative survival factor for severe COVID-19, further emphasizing the prominent role of IFN in SARS-CoV-2 pathogenesis and clearance [21] [22] [23] [24] . Importantly, IFNmediated effects may also be detrimental to COVID-19 outcomes. For example, disruption of the lung epithelium by type III IFN-dependent processes has been hypothesized to expose patients to secondary infections by opportunistic bacteria 25 .

Taken together, while IFN and the downstream expression of ISGs can functionally restrict SARS-CoV-2, the site(s), cell types, amount, and timing of IFN production and response play critical roles in COVID-19 pathogenesis and outcomes.

The specific mechanisms by which IFN restricts SARS-CoV-2 have not been fully characterized. To date, of the hundreds of ISGs, only a handful have been found to restrict SARS-CoV-2 infection in different in vitro systems. Lymphocyte antigen 6 complex, locus-E (LY6E) was identified as a SARS-CoV-2 restriction factor in ISG ectopic cDNA expression screens 26 . A transposon-mediated screen identified the MHC-II invariant chain CD74 as a block to SARS-CoV-2 entry 27 . In addition, overexpression of BST2 (encoding Tetherin), an anti-HIV effector that prevents the release of nascent virions 28 , has also been found to restrict SARS-CoV-2 by impeding virion release 29 . TRIM25, an interferon-induced E3 ubiquitin ligase that enhances antiviral responses downstream of RIG-I, has been shown to interact specifically with SARS-CoV-2 RNA and thereby reduce infection 30 .

Studies evaluating the antiviral potential of individual ISGs are often implemented through the ectopic expression of ISG cDNA libraries followed by viral challenge to screen for those ISGs that confer resistance 3, 4, 29, 31, 32 . While effective in identifying the antiviral potential of many ISGs, arrayed ISG cDNA screens are not without drawbacks including limited throughput, technically demanding cloning and validation of individual expression constructs, and high costs. Ectopic cDNA overexpression can be prone to artifactual expression patterns or functions 33, 34 . In addition, cDNA expression screen libraries typically include only one isoform per gene, and therefore may overlook isoform-specific antiviral activities, as have been described for many ISGs 27, [35] [36] [37] [38] [39] .

CRISPR-activation (CRISPRa), in which guide RNA (gRNA)-directed endonuclease-deficient Cas9 along with transcriptional activators are targeted to a gene of interest (GOI) to induce expression, offer an alternative to cDNA ectopic expression screens. Advantages include easy to produce gRNA libraries, physiologically relevant expression levels, and multiplexing capabilities 40 . Gene transcription is initiated from endogenous promoters, enabling the expression of multiple gene isoforms. Studies in which genome-wide libraries of activating gRNAs uncovered important host factors in viral infection systems provide an important proof of concept to the characterization of ISGs using CRISPRa 41, 42 . Importantly, two recent preprints report genome-wide CRISPRa screens for genes with antiviral potential against SARS-CoV-2 43, 44 .

Here, we report an ISG-focused CRISPRa screen to identify ISGs that modulate SARS-CoV-2 infection in lung epithelial cells. To mitigate the potential antiproliferative and/or proapoptotic effects of certain ISGs that could impact their library representation, we engineered a Doxycycline (Dox)-inducible CRISPRa system that enables precise temporal control of ISG induction. Using a pooled screen strategy, we tested more than 400 ISGs for effects on SARS-CoV-2 infection in both wildtype cells and isogenic cells engineered to be insensitive to IFN. High ranking antiviral ISG hits included SARS-CoV-2 restriction factors previously identified in recent screens (LY6E 26 , CD74 27 , TRIM25 30 and ERLIN1 29 ). We identified and validated antiviral roles for additional ISGs such as CTSS (Cathepsin S). We also identified OAS1 (2′-5′-oligoadenylate synthetase 1) as a SARS-CoV-2 restriction factor capable of inhibiting viral infection and the generation of progeny virus. Taken together, our findings demonstrate the utility of a novel inducible CRISPRa platform for antiviral genetic screens and identify multiple ISGs capable of restricting SARS-CoV-2.

We developed an optimized platform for pooled, positive selection ISG screens by adapting the well-established SunTag CRISPRa technology 45 , and engineering it into A549 lung adenocarcinoma cell lines, a widely employed model for respiratory virus infection [46] [47] [48] . First, we reasoned that the antiproliferative and/or proapoptotic properties of some ISGs 49 could affect their relative representation in pooled libraries prior to infection experiments and/or independent of potential effects on virus susceptibility. To mitigate these effects, we reengineered the transcriptional transactivator component construct of the SunTag CRISPRa system to allow for Doxycycline (Dox)-inducible expression, and thereby Dox-inducible regulation of gRNA-targeted gene expression ( Fig. 1A) . Next, we expected that IFN secretion in response to infection, with corresponding autocrine and paracrine induction of broad ISG expression throughout cultures, could interfere with assessments of individual CRISPRa-induced ISG effects within different cells in pooled screen experiments. Therefore, we transduced the modified SunTag components into A549 ΔSTAT1 cell lines defective in their capacity to respond to IFN (A549 ΔSTAT1 -SunTag 50 ), as well the wildtype A549 cell line (A549-SunTag, intact IFN response) from which they were derived. After selecting and expanding dual antibioticresistant single cell clones, we evaluated their capacity for Dox-inducible, guide RNA (gRNA)-targeted gene expression. We transduced A549-SunTag and A549 ΔSTAT1 -SunTag cell lines with lentiviral constructs expressing gRNAs targeting the promoter region of MX1, a well-characterized ISG that restricts multiple viruses 51, 52 , as well as with non-targeting gRNA (NTG) controls. Following puromycin selection of gRNA-transduced cells, cultures were treated with Dox for 48 hours, and MX1 mRNA expression was assessed by quantitative RT-PCR (qRT-PCR). In both A549-SunTag and A549 ΔSTAT1 -SunTag genotypes, Dox induced robust increases in MX1 mRNA levels in cells expressing MX1 gRNAs (Fig. 1B) , while Dox treatment of cells expressing NTG or nontransduced cells exhibited minimal changes in MX1 gene expression. Comparison to cells pretreated with IFNα2b indicated that CRISPRa induced MX1 mRNA expression to similar levels ( Fig. 1B) . Interestingly, baseline levels of MX1 mRNA exhibited higher Ct values in A549 ΔSTAT1 cells compared to their wildtype counterparts, resulting in greater fold change values for MX1 expression upon Dox treatment ( Fig. 1B-C) . This suggests that, even in the absence of exogenous IFN stimulation, some ISGs exhibit some level of constitutive STAT1-dependent transcription.

Next, we evaluated the functional antiviral capacity of an ISG in our CRISPRa cells.

A549-SunTag and A549 ΔSTAT1 -SunTag cells, transduced and selected to express MX1 gRNA, were treated with Dox and infected with a GFP-encoding Indiana vesiculovirus (VSV-GFP 53 ). Flow cytometry analysis, using GFP expression as a marker for productive infection, demonstrated a near-complete block of infection in Dox On A549-SunTag cells with active MX1 expression; Dox Off cells and cells expressing an NTG were not protected ( Fig. 1D-E) . While we also observed a protective effect in A549 ΔSTAT1 -SunTag, a considerable fraction of cells remained susceptible to infection. These results indicate that Dox-inducible, CRISPRa-mediated ISG expression can effectively restrict viral infection.

Furthermore, the restriction of VSV-GFP infection by MX1 is enhanced by additional, STAT1-dependent factors likely elicited by IFN production, further highlighting the utility of a STAT1-deficient screening platform for assessing the antiviral activity of individual ISGs. In sum, we have established a functional, Dox-regulated CRISPRa system in isogenic cell lines with either intact or deficient IFN responses that effectively restricts viral infection upon induced expression of antiviral ISGs.

To identify ISGs that restrict SARS-CoV-2, we conducted pooled gene activation screens in our engineered CRISPRa A549-SunTag lines. Our general screening strategy was to evaluate the potential of hundreds of individual ISGs to confer resistance to the cytopathic effects of SARS-CoV-2. We began by introducing ACE2 expression into A549-SunTag and A549 ΔSTAT1 -SunTag cells to enable productive SARS-CoV-2 infection of our To identify ISGs with an effect on SARS-CoV-2 CPE in A549-SunTag ACE2 or A549 ΔSTAT1 -SunTag ACE2 cells, we applied a stringent series of filters for gRNAs differentially enriched by SARS-CoV-2 infection (infection status adjusted p value < 0.1), while accounting for Dox effects (interaction adjusted p value < 0.1). We detected 6 ISGs (A549-SunTag ACE2) and 24 ISGs (A549 ΔSTAT1 -SunTag ACE2) as "antiviral" (i.e. Interestingly, when comparing ISG hits between A549-SunTag ACE2 and A549 ΔSTAT1 -SunTag ACE2 screens (Fig. 2D) , we found only a single common antiviral hit across genotypes: OAS1 (encoding 2′-5′-oligoadenylate synthetase 1). Not surprisingly, our results suggest that STAT1-dependent transcription, perhaps in response to endogenous IFN production in A549-SunTag ACE2 screens, modulates detection of CRISPRainduced ISG effects on SARS-CoV-2. As illustrated in Figure 2D , many of our ISG hits were similarly selected in both STAT1 genotypes (i.e. both antiviral or both proviral), but failed to clear significance thresholds in one of the screens.

In sum, in conducting focused CRISPRa ISG screens for cell viability effects in A549 cells infected with SARS-CoV-2, we detected several previously known SARS-CoV-2 restriction factors as well as identified new candidate ISGs with putative anti-SARS- -Log 10 Adjusted.P-value Z score Dox On infected vs Dox On mock Hits -Log 10 Adjusted P value 

0.5 1 PDGFRL LY6E OAS1 CD74 CDK18 ZNFX1 BCL2L14 SQLE TNFRSF10A TREX1 TF MTHFD2L TAP1 CDKN1A TNFSF10 CTSL -1 0 1 Gene β score SARS2 infected IFIT2 PIM3 B4GALT5 TLR7 SLC15A3 JAK2 OAS1 CTSS SERPINE1 ERLIN1 CLEC4D GCA A D - GBP1 NCOA7 PLIN2 SAMD9 MCL1 IFITM1 TRIM21 CHMP5 VEGFC GBP3 -1 0 1 Gene β score SARS2 infectedMTHFD2L TAP1 CDKN1A TNFSF10 CTSL 0 1 IFIT2 PIM3 B4GALT5 TLR7 SLC15A3 JAK2 TRIM25 OAS1 CTSS SERPINE1 ERLIN1 CLEC4D GCA ADPRHL2 UNC93B1 GBP1 NCOA7 PLIN2 SAMD9 WHAMM MCL1 IFITM1 TRIM21 CHMP5 VEGFC GBP3

To confirm hits identified in the pooled screens, we first conducted "single gene" validation experiments with the CRISPRa system for a subset of candidate antiviral ISGs. Interestingly, the apparent antiviral effect conferred by cDNA expression of CD74 was weaker than that observed in corresponding CRISPRa gRNA-directed expression experiments (Fig. 4A, compared to Fig. 3A ). Although these differences could be due to dissimilar expression levels in the CRISPRa and cDNA systems, they might also be explained by isoform-specific effects; the anti-SARS-CoV-2 effects of CD74 have been assigned to the p41 isoform 27 that can be expressed in CRISPRa experiments, while the "canonical" isoform p45 (Uniprot identifier P04233-1) was tested in our cDNA experiments. In sum, these experiments offer further validation of the antiviral activity of ISG screen hits against SARS-CoV-2. 

OAS1 was detected as an antiviral ISG hit in both A549-SunTag and A549 ΔSTAT1 -SunTag screens (Fig. 2) and validated in targeted CRISPRa experiments at both time points tested (Fig. 3) . transduced with NTG were readily eliminated by infection (Fig. 5B ). In addition to protective effects on cell viability, we also tested the direct impact of OAS1 expression on the propagation of SARS-CoV-2. To minimize the effects of endogenous IFN production, we infected Dox Off and Dox On A549 ΔSTAT1 SunTag ACE2 cells expressing OAS1 or NTG gRNAs and quantified viral titers by plaque assay over time. Activation of OAS1 expression resulted in a significant decrease in the production of viral progeny, indicating that OAS1 activity functionally restricts SARS-CoV-2 infection (Fig. 5C ).

OAS1 isoforms have been shown to differ in their antiviral activities 36 Fig. 3B ). These results support potential roles for many ISGs, including many with established antiviral activities, in IFN effects on cell viability and proliferation.

Here, we report a CRISPRa strategy for pooled screens of IFN-induced antiviral effectors, and employed this approach to identify ISGs that restrict SARS-CoV-2 cytopathogenicity. Screen results included previously described SARS-CoV-2 restriction factors, as well as multiple additional candidate ISGs with antiviral activity against SARS- Some ISGs suppress cell proliferation or promote apoptosis 1 . These molecular 

Oligoadenylate synthetase family members are broadly acting ISGs important for innate antiviral defense against multiple viruses 66 . RNaseL, the downstream effector of 24.

2%

O n _ S A R S 2 _ 2 _ E X P 1 0 500,000 1,000,000 1,500,000 2,000,000 2,500,000 3,000,000 3,500,000 In analyses for antiproliferative/proapoptotic ISGs, hits were selected as adjusted p value less than 0.1 for the Dox status coefficient (Dox Off mock infected vs Dox On mock infected), and b score were multiplied by -1 to facilitate enrichment/depletion interpretation within the required model syntax.

Cloning of individual gRNAs into CROP-seq-opti vectors was performed as previously described 96, 97 

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We thank Mayte Suarez-Farinas for advice on statistics and experimental design, and Thomas Moran, Center for Therapeutic Antibody Discovery at the Icahn School of Medicine at Mount Sinai, for kindly providing anti-SARS-CoV Nucleocapsid antibody. We thank Meike Dittmann for A549 ΔSTAT1 cell lines, Benjamin R. tenOever and Skyler Uhl for technical assistance with plaque assays, and Michael A. Schotsaert for flow cytometry support. We also thank Randy Albrecht for BSL3 facility management and support. We thank Dusan Bogunovic for discussion, helpful advice, and critical reading of the manuscript. We thank all members of the Rosenberg lab for advice and support. This work was supported in part by NIH grants R01 AI151029 and U01 AI150748, and funds from the Department of Microbiology, Icahn School of Medicine at Mount Sinai. Research reported in this paper was also supported by the Office of Research Infrastructure of the National Institutes of Health under award numbers S10OD018522 and S10OD026880.The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders, including the National Institutes of Health.

Authors declare no competing interests.

Sequence data files from inducible CRISPRa ISG screens are available from the NCBI Sequence Read Archive (SRA), accession number pending.