Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection

When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.

negative charge that is able to interact electrostatically with the basic residues that are exposed by viral surface glycoproteins. This allows viruses to increase their concentration at the cell surface and thus the possibility to interact with their specific entry receptor (2). For instance, alphaviruses, such as Semliki Forest virus (SFV) and Sindbis virus (SINV), are enveloped positive-strand RNA viruses that contain two glycoproteins at the envelope, namely, the proteins E1 and E2. E2 is involved in the interaction of the virus particle with the cell surface (3,4), while E1 serves in the fusion process (5).
Once the virus is inside the cell, the replication of the viral genome represents another critical step for triggering the antiviral immune response. Double-stranded RNA (dsRNA) is a ubiquitous pathogen-associated molecular pattern (PAMP) recognized by the cellular machinery, which can arise as a replication intermediate for viruses with an RNA genome or from convergent transcription for DNA viruses (6). In mammals, dsRNA recognition is driven by specific receptors, including the cytoplasmic RIG-like receptors (RLRs) and endosomal Toll-like receptors (TLRs) (7). Sensing of dsRNA by these receptors results in the activation of a complex signaling cascade leading to the production of type I interferon (IFN), which in turn triggers the expression of IFN-stimulated genes (ISGs) and the establishment of the antiviral state (8). The ultimate outcome of this vertebrate-specific antiviral response is translation arrest and cell death by apoptosis (9).
The revolution brought by the discovery of the CRISPR-Cas9 technology has provided biologists with an invaluable tool for editing the genome at will and easily performing individual gene knockout (KO) (10). This technique is perfectly suited to perform genome-wide screens in a relatively fast and easy-to-implement manner, especially when the readout is based on cell survival. For this reason, numerous CRISPR-Cas9 loss-of-function screens have been performed based on cell survival after infection with different viruses (11)(12)(13). These approaches allowed the identification of novel virus-specific as well as common factors involved in antiviral defense mechanisms or in cellular permissivity to virus infection.
Here, we chose to take advantage of the fact that dsRNA is almost always detected in virus-infected cells (6) and is a potent inducer of apoptosis to design a genome-wide screen aimed at identifying host genes that when edited resulted in increased cell survival to dsRNA and viral challenge. To this aim, we performed a CRISPR-Cas9 screen based on cell survival in HCT116 cells after either cationic lipid-based transfection of an in vitro-transcribed long dsRNA or infection with the model alphavirus SINV, which replicates via a dsRNA intermediate.
Our results indicate that genes involved in limiting attachment and therefore entry, be it of the synthetic dsRNA or SINV, are vastly overrepresented after selection. We validated two genes of the heparan sulfate pathway (namely, SLC35B2 and B4GALT7) as being required for dsRNA transfectability and SINV infectivity. We also identified and characterized COG4, a component of the conserved oligomeric Golgi (COG) complex, as a novel factor involved in susceptibility to dsRNA and viral-induced cell death linked to the heparan sulfate biogenesis pathway.

RESULTS
Genome-wide CRISPR-Cas9 screen based on cell survival upon dsRNA transfection identifies factors of the heparan sulfate pathway. In order to identify cellular genes that are involved in the cellular response to dsRNA, which culminates with cell death, we performed a CRISPR-Cas9 genome-wide loss-of-function screen in the human colon carcinoma cell line HCT116. This cell line is highly suitable for CRISPR-Cas9 genetic screening procedures (14) and can be easily infected with SINV with visible cytopathic effects at 24 and 48 hours postinfection (hpi) (see Fig. S1A in the supplemental material). Moreover, transfection of an in vitro-transcribed 231-bp-long dsRNA by a cationic lipid-based transfection reagent in HCT116 cells led to strong cell death at 24 and 48 hours posttreatment (hpt) (Fig. S1B).
We generated a Cas9-expressing HCT116 monoclonal cell line (Fig. S1C) that we stably transduced with the human genome-wide lentiviral Brunello library composed of 76,441 single guide RNAs (sgRNAs) targeting 19,114 genes, as well as about 1,000 nontargeting sgRNAs as controls (15). We then applied a positive selection by lipofection of 30 million transduced cells per replicate with the synthetic long dsRNA, and we collected the surviving cells 48 h later. In parallel, the same initial amount of stably transduced cells was left untreated as a control (input) for each replicate (Fig. 1A). DNA libraries from the input samples were generated, sequenced, and quality checked. In particular, we verified the sgRNA coverage by observing the presence of the 4 guides per gene for 18,960 genes (99.2% of the genes) and 3 sgRNAs per gene for the remaining 154 genes (0.2% of the genes) (see Data Set S1 in the supplemental material).
Using the MAGeCK software (16), we assessed the normalized read count distribution of the control and dsRNA-treated biological triplicates, which, despite a quite homogenous sgRNA distribution, showed the presence of few outliers upon selection (Fig. 1B). We identified eight genes that were significantly enriched with a false discovery rate lower than 1% (FDR1%). Among those genes, four belonged to the heparan sulfate biosynthesis pathway (namely, SLC35B2, B4GALT7, EXT1, and EXT2) and three were components of the conserved oligomeric Golgi complex (namely, COG3, COG4, and COG8) ( Fig. 1C; see Data Set S2 in the supplemental material). In particular, all four sgRNAs targeting each of the SLC35B2, B4GALT7, and COG4 genes were enriched upon dsRNA selection (Fig. S1D).
Heparan sulfate is a linear polysaccharide that is covalently attached to core proteins in proteoglycans (PGs) on the cell surface (for review, see reference 17). Among many properties, HS plays a role in binding protein ligands and as a carrier for lipases, chemokines, and growth factors (17,18), but also as a viral receptor (19). HS biosynthesis takes place in the Golgi, where most of the biosynthetic enzymes are anchored to the Golgi membrane (20).
We first validated the resistance phenotype to dsRNA of SLC35B2 and B4GALT7, the two top hits identified in the screen (Fig. 1C, Fig. S1D), by generating two individual knockout clones for each gene by CRISPR-Cas9 editing in HCT116cas9 cells (see The observed resistance to dsRNA in the mutants could occur at many different steps, namely, dsRNA liposome attachment and entry, recognition, induction of the IFN pathway, or apoptosis. To test whether the first step was affected, we employed a nucleic acid delivery method that was not based on cationic lipid transfection. In particular, we used nucleofection (an electroporation-based transfection method) to introduce long dsRNAs into HCT116 cells, and we showed that this approach restored cell death in SLC35B2 and B4GALT7 knockout cells (Fig. 1D, right part of the graph). In addition, we performed liposome-based transfection of an in vitro-transcribed Cy5labeled dsRNA in SLC35B2 and B4GALT7 KO cells and assessed the Cy5 fluorescence at 48 h posttransfection by fluorescence-activated cell sorter (FACS) analysis ( Fig. 1E and F). Although the number of Cy5 positives (Cy5ϩ) cells was not significantly different in the B4GALT7 KO clones and was only slightly lower in the SLC35B2 KO cells than in wild-type (WT) cells (Fig. 1E), we observed a significant reduction of at least 80% of the median Cy5 fluorescence in both B4GALT7 and SLC35B2 KO cells relative to the control (Fig. 1F), thereby indicating a significant drop in the number of transfected Cy5-labeled RNA molecules per cell.
We also confirmed that liposome-based transfection of nucleic acids, such as plasmidic DNA, was impaired in SLC35B2 and B4GALT7 KO cells by transfecting a green fluorescent protein (GFP)-expressing plasmid using Lipofectamine 2000 in wild-type or knockout cells (see Fig. S3A, left, and Fig. S3B in the supplemental material). Nonetheless, GFP expression could be restored in all cell lines upon nucleofection (Fig. S3A, right). To establish whether impairment of the HS synthesis is directly linked to a defect in dsRNA entry and increased cell survival, we measured the extracellular HS levels in SLC35B2 and B4GALT7 KO cells. We measured a substantial reduction of the extracellular HS staining, as assessed by FACS measurement of two independent SLC35B2 and B4GALT7 KO clones compared with HCT116 wild-type cells (Fig. 1G). To confirm the importance of HS at the cell surface for liposome-based transfection, we mimicked the HS-defective phenotype by removing extracellular HS in parental HCT116cas9 cells either enzymatically (with heparinase) or chemically (with sodium chlorate [NaClO 3 ]) ( Fig. S3C and D). We tested the transfectability of a GFPexpressing plasmid by measuring either the relative number of GFP-positive cells or the relative median of GFP intensity of fluorescence by FACS analysis. Although the relative number of GFP-positive cells was not significantly reduced by heparinase treatment (Fig. S3C), it caused a reduction in GFP intensity in HCT116cas9-treated cells, thereby recapitulating the GFP plasmid lipofection defect observed in SLC35B2 and B4GALT7 KO (Fig. S3D). Moreover, this effect correlated with the reduction of extracellular HS by enzymatic treatment quantified by FACS (Fig. S3E), which demonstrated that extracellular HS are crucial for transfection by lipofection. In the case of NaClO 3 treatment, despite the reduction in both the relative number of GFP-positive cells (Fig. S3C) and the relative median of GFP intensity of fluorescence ( Fig. S3D) compared with the control, we could not observe a correlation with a decrease in overall extracellular HS (Fig. S3E). This result could be due to the fact that while a mix of heparinase I and III removes every kind of extracellular heparan sulfates, NaClO 3 impairs only the O-sulfation (21).
Taken together, our results show that knocking out SLC35B2 and B4GALT7 results in reduced levels of extracellular HS, which in turn impairs liposome-based transfectability of HCT116 cells. Moreover, the validation of these two top hits indicates that other candidates might be suitable for further analysis and may also have an impact on the dsRNA resistance phenotype.
COG4 is involved in dsRNA-induced cell death partly via the heparan sulfate pathway. Among the significant hits of our genome-wide screen were proteins related to the COG complex, namely, COG4, COG3, and COG8. The COG complex is a heterooctameric complex containing 8 subunits (COG1 to COG8) interacting with numerous proteins mainly involved in intra-Golgi membrane trafficking, such as vesicular coats, Rab proteins, and proteins involved in the SNARE complex (22,23). This interaction with the trafficking machinery is crucial for the proper functionality of the Golgi apparatus, and mutations in the COG complex result in severe cellular problems, such as glycosylation defects (24)(25)(26)(27), which are due to mislocalization of recycling Golgi enzymes (28,29).
Since we retrieved three out of the eight COG family members in our CRISPR-Cas9 screen suggesting their importance in dsRNA-induced cell death, we tested the effect S. pyogenes Cas9 protein were transduced with the lentiviral sgRNA library Brunello (MOI, 0.3). Thirty million transduced cells per replicate were selected with 1 g/ml puromycin to obtain a mutant cell population to cover at least 300ϫ the library. Selective pressure via synthetic long dsRNA (1 g/ml) was applied to induce cell death (in red). DNA libraries from input cells and cells surviving the dsRNA treatment as three independent biological replicates were sequenced on an Illumina HiSeq 4000 instrument. Comparisons of the relative sgRNA boundance under the input and dsRNA conditions were done using the MAGeCK standard pipeline. (B) Median normalized read count distribution of all sgRNAs for the input (in black) and dsRNA (in red) replicates. (C) Bubble plot of the candidate genes. Significance of robust rank aggregation (RRA) score was calculated for each gene in the dsRNA condition compared with that of input using the MAGeCK software. The number of enriched sgRNAs for each gene is represented by the bubble size. The gene ontology pathways associated with the significant top hits are indicated in orange and green. (D) Viability assay. Cells were transfected (80,000 cells; 1 g/ml) or nucleofected (200,000 cells; 400 ng) with synthetic long dsRNA, and cell viability was quantified 24 h (nucleofection) or 48 h (transfection) posttreatment using PrestoBlue reagent. The average of at least three independent biological experiments Ϯ SD is shown. One-way ANOVA analysis; *, P Ͻ 0.05. (E, F) Cy5-labeled dsRNA (80,000 cells; 1 g/ml) was transfected into HCT116cas9, B4GALT7#1 and 2, and SLC35B2#1 and #2 cells; and Cy5 fluorescence was quantified using FACS (10,000 events). The relative number of the Cy5-positive (Cy5ϩ) cells (E) and the relative median of Cy5 intensity of fluorescence (F) compared to those of HCT116cas9 cells are shown. The average of three independent biological experiments Ϯ SD is shown. Paired t test analysis; *, P Ͻ 0.05. (G) Quantification of extracellular heparan sulfates. FACS analysis of HCT116 control or KO clones stained with the HS-specific antibody 10E4 (in red) compared to unstained samples (in blue) (10,000 events). One representative experiment out of three is shown. of their inactivation by CRISPR-Cas9. As COG4 is the most enriched COG gene in our screen, we generated a polyclonal COG4 KO HCT116cas9 cell line and validated their dsRNA resistance phenotype resulting in an increase survival in response to synthetic dsRNA transfection (see Fig. S4A in the supplemental material). We also observed a reduction in the relative number of Cy5-positives (Cy5ϩ) cells and the median Cy5 fluorescence in COG4 KO cells relative to the control by FACS analysis (Fig. S4B and C).
To further confirm the involvement of the COG complex, we also tested the effect of dsRNA transfection in previously generated HEK293T KO COG3, COG4, and COG8 cells ( Fig. 2A) (30). Interestingly, while COG8 mutants did not display a significant survival phenotype in response to dsRNA lipofection, COG3 and COG4 KO HEK293 cells did. In addition, the survival phenotype could be complemented by stable expression of a COG4-GFP construct compared with COG4 KO cells ( Fig. 2A). Moreover, although we could not detect a decrease in the relative number of Cy5-positive (Cy5ϩ) cells in COG4 KO cells relative to the controls (Fig. 2B), the median Cy5 fluorescence in COG4 KO cells was significantly reduced compared with both HEK293T and COG4-rescued cells (Fig. 2C), thereby indicating a significant decrease in the number of transfected Cy5-labeled RNA molecules per cells.
In agreement, dsRNA accumulation appeared to be significantly reduced, but still present, in HEK293T COG4 KO cells compared with control cells, as determined by reverse transcriptase quantitative PCR (RT-qPCR) analysis of dsRNA isolated from cells 24 h after transfection (Fig. 2D), and this correlated with reduced IFN-beta accumulation in HEK293T KO COG4 cells compared with control cells (Fig. 2E).
These results indicated that dsRNA transfectability was strongly reduced but not completely impaired in the absence of COG4 and that dsRNA could be still detected in COG4 KO cells in order to activate type-I IFN response.
We confirmed the reduced internalization of dsRNA in COG4 mutant cells by transfecting rhodamine-labeled poly(I·C), a synthetic dsRNA analog, in HEK293T COG4 KO or WT cells (Fig. 2F) and by counting the number of poly(I·C) foci per cell at 6, 12, and 24 h posttransfection (Fig. 2G). We could observe a significant reduction in rhodamine-positive foci in HEK293T COG4 KO during the time course, suggesting a defect in dsRNA internalization, which could explain the increased survival phenotype.
In order to assess whether the COG4 KO survival phenotype was associated with a defect in the heparan sulfate pathway, we stained extracellular HS and measured the HS expression by FACS analysis. We observed a decrease of extracellular HS in KO COG4 cells compared with control cells (WT and rescued), which demonstrated that the COG complex is related to the HS biosynthesis pathway (Fig. S4D).
The reduction in extracellular HS could correlate with a decrease in transfectability and explain the survival phenotype in KO COG4 cells. Surprisingly, however, lipofection of a GFP-expressing plasmid indicated that HEK293T COG4 KO cells are still transfectable with a plasmid DNA compared with control cells, as observed by FACS analysis (Fig. S4E and F).
Altogether, these findings indicate that the COG complex is involved in HS biosynthesis and that removal of COG4 results in a lower accumulation of HS at the cell surface, which most likely translates to a reduced transfectability of dsRNA. However, as opposed to the observations in SLC35B2 or B4GALT7 KO cells, the cells are still transfectable with a plasmid DNA and, although to a lower extent, with dsRNA. Interestingly, the increased cell survival phenotype of COG4 KO cells upon dsRNA transfection does correlate with a reduced, but still measurable, IFN-␤ production.
Cell survival-based genome-wide CRISPR-Cas9 screen identifies COG4 as a permissivity factor to SINV. SINV is a small enveloped virus with a single-stranded RNA genome of positive polarity. The virus belongs to the Togaviridae family, Alphavirus genus, and is considered the model for other medically important viruses, such as chikungunya virus (CHIKV) and Semliki Forest virus (SFV). During its infectious cycle, SINV produces dsRNA as a replication intermediate and induces cytopathic effects in mammalian cells, leading to cell death within 24 to 48 h postinfection (31).
In order to identify host genes that are related to SINV-induced cell death and infection, we performed a CRISPR-Cas9 knockout screen in HCT116cas9 cells, which are susceptible to this virus ( Fig. 3A and Fig. S1). After transduction with the CRISPR lentiviral genome-wide knockout library, puromycin-resistant HCT116 cells were infected with SINV-GFP at a multiplicity of infection (MOI) of 0.1 and selected for cell survival. Using the MAGeCK software (16), we assessed the normalized read count distribution of the control and SINV-infected biological triplicates, which, despite a quite homogenous sgRNA distribution, showed the presence of few outliers upon selection (Fig. 3B). We identified two genes that were significantly enriched with a false , and rescued HEK293T cells were infected with SINV GFP for 24 and 48 h at an MOI of 1, and the supernatant was collected in order to measure viral production. The fold change in titer relative to HEK293T arbitrarily set to 1 is shown. The average of three independent biological experiments Ϯ SD is shown. Paired t test analysis; *, P Ͻ 0.05. discovery rate lower than 25% (FDR25%), notably SLC35B2 and B4GALT7 (see Data Set S3 in the supplemental material, Fig. 3C). Genes of the heparan sulfate pathway have been previously found in genome-wide CRISPR-Cas9 loss-of-function studies looking for factors involved in the accumulation of viruses, such as influenza, Zika, and chikungunya viruses (11,32,33). Interestingly, among the top-ranking hits, we retrieved COG4, which was not previously associated with SINV infection (Fig. 3C).
To validate the involvement of COG4 during SINV infection, we infected HEK293T, COG4 KO, or COG4 KO-rescued HEK293T cells with SINV and measured cell viability at 24, 48, and 72 hpi. The cell viability assay revealed that the COG4 KO cells were less sensitive at early times points of SINV infection (24 and 48 hpi), but this tendency disappeared at 72 hpi (Fig. 3D). In agreement, the determination of viral titer by plaque assay showed that COG4 KO HEK293T cells produced significantly fewer infectious viral particles than HEK293T or COG4-rescued cells at 24 hpi but not at 48 hpi, underlining a possible delay in the infection and virus-induced cell death (Fig. 3E). We also observed that GFP accumulated to lower levels in COG4 KO than in WT cells both at 24 and 48 h postinfection (hpi) (Fig. S5A). Finally, we noticed that the reduced viral production in COG4 KO cells was associated with a reduced accumulation of viral dsRNA in the cytoplasm when we infected WT or COG4 KO cells with SINV-GFP and performed immunostaining with the anti-dsRNA J2 antibody 24 hpi (Fig. S5B). Overall, our results indicate that COG4 expression is needed for an efficient SINV infection and that its absence can delay the infection, thereby increasing cell survival in COG4 KO cells.

DISCUSSION
Several CRISPR-Cas9 screens aimed at identifying factors required for infection by specific viruses have been described in the literature, but to our knowledge, none has been designed to look at the effect of the only common factor between all those viruses, i.e., dsRNA. Here, we used the Brunello sgRNA lentiviral library to screen for genes involved in HCT116 cell survival to synthetic dsRNA transfection and to SINV infection. This screen allowed us to identify components of the heparan sulfate biosynthesis pathway and of the COG complex that are critical host factors in the cellular response to both long dsRNA transfection and SINV infection challenges. It has been reported that cell survival-based CRISPR screens for viral host factors are biased toward genes linked to the initial steps of the infection and even more so to viral entry (11,34). Thus, in our case, HS is a well-known factor required for SINV entry due to virus adaptation to cell culture (35). We also retrieved genes of the HS pathway in our dsRNA-based screen, and we confirmed the importance of extracellular HS for dsRNAinduced toxicity. This is mostly due to a decrease of cell transfectability when HSs are missing, which is linked to the fact that the polyplexes used for transfection are positively charged and can interact electrostatically with glycosaminoglycans (36,37). Our work also addresses limitations of survival-based CRISPR-Cas9 screens. Thus, in this study, the selection pressure was too strong to allow the identification of genes intervening after the entry step, thereby making the screen less sensitive. Adjusting the selection procedure by reducing the concentration of dsRNA or increasing the duration of treatment may allow the identification of hits not solely implicated in the transfection process but also the innate response of the cells to either dsRNA or the transfection agent. Alternatively, new strategies should be designed to overcome this problem, such as using fluorescent-based cell sorting in order to be less stringent in the selection.
In addition to the HS pathway, we identified members of the COG complex, and more specifically COG4, as factors involved in dsRNA transfection and SINV infection. Loss-of-function COG4 mutant cells show a dsRNA-resistant phenotype as well as a reduction in extracellular HS expression, which is similar to previously published reports for other COG proteins (33,38). Surprisingly, even if the removal of COG4 expression results in a defect in the HS pathway, we were still able to transfect the KO COG4 cell line either with a plasmid encoding GFP or, although to a lesser extent, dsRNA. In addition, the dsRNA molecules that are able to enter into COG4 KO HEK293T cells are still sufficient to induce the IFN-␤ mRNA production, indicating that the innate immune response is still functional in these mutant background. Nonetheless, cell death induced by dsRNA appears to be lower in COG4 KO cells, most likely due to a delay in the infection.
Future work will be needed to assess whether this phenotype upon SINV infection is correlated only with a defect in HS biogenesis in COG4 mutants or with other functions of COG4. The increased cell survival to synthetic dsRNA transfection and viral infection of COG4 KO cells compared to WT cells opens several interesting perspectives. Indeed, since the COG complex is related to glycosylation and membrane trafficking (23,25,39,40), deficiency in one or more of its components could potentially lead to a glycosylation and/or subcellular localization defect of components of the innate immune response or of the apoptosis pathway, although this possibility remains to be formally proven. The difference of transfectability of plasmid DNA and dsRNA in COG4 KO cells is also intriguing and could indicate that different kinds of nucleic acids do not necessarily use the exact same routes to enter the cells upon liposome-based transfection. Finally, there could be other defects linked to COG deficiencies (39, 41) that could account for our observations, and elucidating those defects will require further work. It is particularly interesting that COG3 and COG4 knockout cells display a dsRNA-induced cell death resistance phenotype, while COG8 mutants do not. This finding implies that only part of the COG complex is involved in dsRNA uptake.
In conclusion, our work uncovered COG4 as a new player in HS production, which is required for both SINV infection and dsRNA transfection. These results also highlight that synthetic dsRNA is a powerful tool for identifying novel key pathways of the cellular response to RNA viruses.
SINV wild type (SINV WT) or SINV expressing the green fluorescent protein (SINV GFP) were produced as previously described (44) in BHK-21 cells. In SINV GFP, the promoter of SINV subgenomic RNA was duplicated and inserted at the 3= extremity of the viral genome, and the GFP sequence was then inserted after this new promoter. Cells were infected with either SINV WT or SINV GFP at an MOI of 10 Ϫ1 , and samples were harvested at 24 or 48 hours postinfection (hpi).
Standard plaque assay. Ten-fold dilutions of the viral supernatant were prepared. Fifty-microliter aliquots were inoculated onto Vero R cell monolayers in 96-well plates for 1 hour. Afterward, the inoculum was removed, and cells were cultured in 2.5% carboxymethyl cellulose for 72 h at 37°C in a humidified atmosphere of 5% CO 2 . Plaques were counted manually under the microscope. For plaque visualization, the medium was removed and cells were fixed with 4% formaldehyde for 20 min and stained with 1ϫ crystal violet solution (2% crystal violet [Sigma-Aldrich], 20% ethanol, and 4% formaldehyde). J2 immunostaining. HEK293T or KO COG4 HEK293T cells were plated onto a Millicell EZ slide (Millipore) and were infected with SINV at an MOI of 0.1 for 24 h. Cells were fixed with 4% formaldehyde diluted in 1ϫ phosphate-buffered saline (PBS) for 10 min at room temperature (RT), followed by incubation in blocking buffer (0.2% Tween X-100, 1ϫ PBS, and 5% normal goat serum) for 1 h. J2 antibody (Scicons) diluted in blocking buffer at 1:1,000 was incubated overnight at 4°C. Between each step, cells were washed with 1ϫ PBS-0.2% Tween. Secondary antibody goat anti-mouse Alexa 594 (ThermoFisher) diluted at 1:1,000 in 1ϫ PBS-0.2% Tween was incubated for 1 h at room temperature. After 4=,6-diamidino-2-phenylindole (DAPI) staining (1:5,000 dilution in 1ϫ PBS for 5 min), slides were mounted with a coverslip over antifading medium and observed by epifluorescence microscopy using the BX51 (Olympus) microscope with a 40ϫ lens objective.
Generation of HCT116cas9 line. The HCT116cas9 cells, expressing the human codon-optimized Streptococcus pyogenes Cas9 protein, were obtained by transducing the wild-type HCT116 colorectal carcinoma cell line (ATCC CCL-247) with a lentiCas9-BLAST lentiviral vector (no. 52962; Addgene). Briefly, wild-type HCT116 cells were cultured in standard DMEM (Gibco) medium supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml of penicillin-streptomycin (Gibco) at 37°C in 5% CO 2 . The cells were transduced at 80% confluence in a 10-cm tissue culture plate, using 6 ml of lentiviral supernatant supplemented with 4 g/ml of Polybrene (H9268; Sigma) for 6 hours. The transduction medium was replaced with fresh growing medium for 24 h before starting the selection. Transduced HCT116cas9 cells were selected for 10 days and maintained in growing medium supplemented with 10 l/ml of blasticidin (Invivogen).
High-titer lentiviral sgRNA library production. The production of a high-titer human sgRNA Brunello lentiviral library, which contains 4 sgRNAs per gene (15) (no. 73178; Addgene), was performed by transfecting HEK293T cells in five 15-cm tissue culture plates using the polyethyleneimine (PEI) (linear; molecular weight [MW], 25,000; no. 23966-1-A; Polysciences) transfection method (45). Briefly, for each 15-cm plate containing 20 ml of medium, 10 g of sgRNA library, 8 g of psPAX2 (plasmid no. 12260; Addgene), and 2 g of pVSV-G (plasmid no. 138479) diluted in 500 l of 150 mM NaCl were combined with 40 l of PEI (1.25 mg/ml) dissolved in 500 l of 150 mM NaCl. The mix was incubated for 30 minutes at room temperature, and the formed complexes were added dropwise on the cells. After 6 hours, the medium was replaced, and the viral supernatant was collected after 48 hours and after 72 hours. The supernatant was filtered through a 0.45-m polyethersulfone (PES) filter and the viral particles concentrated 100 times using the Lenti-X concentrator (TaKaRa) before storage at Ϫ80°C. The viral titer was established by counting puromycin-resistant colonies formed after transducing HCT116 cells with serial dilutions of the viral stock. HCT116cas9 cells were transduced with lentivirus-packaged Brunello sgRNA library at an MOI of 0.3. The lentiviral library has been sequenced to verify that all the lenti-sgRNA are represented.
Genome-wide CRISPR-Cas9 knockout screens. For each replicate (n ϭ 3), 5 million stably transduced cells/dish were seeded in six 15 cm 2 plates in order to keep a 300ϫ representativity of the sgRNA library. Untreated samples (input) were collected as controls. One day later, cells were either lipofected with 1 g/ml dsRNA-citrine or infected with SINV at MOI of 0.1 and cultured at 37°C and 5% CO 2 . Cells were washed with 1ϫ PBS 48 hours posttreatment, to remove dead cells, and fresh medium was added to surviving clones. Cells were expanded, and all cells were collected 6 days post-dsRNA transfection and 18 days post-SINV infection.
Genomic DNA was isolated by resuspending the cell pellet in 5 ml of resuspension buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 100 g/ml RNaseA), and 0.25 ml of 10% SDS was added and incubated 10 min at RT after mixing. After incubation, the sample was sonicated and incubated 30 min at RT with 10 l of proteinase K (10 mg/ml). A total of 5 ml of a phenol/chloroform/isoamyl alcohol solution was added, followed by a centrifuge step for 60 min at 12,000 ϫ g and 20°C. The upper phase was transferred into a new tube, and 500 l of 3M NaAc and 5 ml of isopropanol was added and incubated overnight at RT, followed by a centrifuge step for 30 min at 20°C and 12,000ϫ g. The pellet was washed using EtOH and dissolved in H 2 O.
Illumina P5-and P7-barcoded adaptors were added by PCR on genomic DNA (gDNA) samples according to the GoTaq protocol (Promega). PCR amplicons were gel purified and sequenced on a HiSeq 4000 instrument (Illumina) to obtain about 30 million reads for each sample. The enrichment of sgRNAs was analyzed using MaGeCK with default parameters (16). The primers used to generate the PCR products are listed in Table S1 in the supplemental material. The results of the dsRNA and SINV screen are available in Data Set S1 and S2, respectively Generation of monoclonal SLC35B2 and B4GALT7 and polyclonal COG4 knockout HCT116 cell lines. The sgRNA expression vectors targeting SLC35B2, B4GALT7, or COG4 (sgRNA sequences selected were the 2 most enriched sgRNA from the Brunello library in the dsRNA screen) genes were produced by annealing the "sense" and "antisense" oligonucleotides (Table S1) at a concentration of 10 M in 10 mM Tris-HCl (pH 8.0) and 50 mM MgCl 2 in 100 l. The mixture was incubated at 95°C for 5 minutes and then allowed to cool down to room temperature. The oligonucleotide duplex thus formed was cloned into the BbsI restriction site of the plasmid pKLV-U6gRNA (BbsI)-pGKpuro2ABFP (no. 62348; Addgene). The lentiviral supernatant from the single transfer vector was produced by transfecting HEK293T cells (ATCC CRL-3216) with the transfer vector psPAX2 packaging plasmid ( no. 12260; Addgene) and the pVSV envelope plasmid (no. 8454; Addgene) in the proportion 5:4:1 using Lipofectamine 2000 (ThermoFisher) reagent according to manufacturer's protocol. Standard DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 U/ml of penicillin-streptomycin (Gibco) was used for growing HEK293T cells and for lentivirus production. One 10-cm plate of HEK293T cells at 70% to 80% confluence was used for the transfection. The medium was replaced 8 hours posttransfection. After 48 h, the medium containing viral particles was collected and filtered through a 0.45-m PES filter. The supernatant was directly used for transfection or stored at Ϫ80°C. A 6-well plate of HCT116cas9 cells at 80% confluence was transduced using 600 l of the lentiviral supernatant (300 l of each lentivirus produced for each duplex) supplemented with 4 g/ml Polybrene (Sigma) for 6 h. The transduction medium was then changed with fresh DMEM for 24 hours, and then the transduced cells were selected using DMEM containing 10 g/ml blasticidin (Invivogen) and 1 g/ml puromycin (Invivogen). Genomic DNA was isolated from individual colonies, and KO clones were screened by PCR (primers in Table S1). The expected WT band for SLC35B2 is 469 bp and the mutant band is 132 bp. For B4GALT7, the WT band is 341 bp and mutant band is 180 bp. For laboratory purposes, the SLC35B2 clones have been generated into HCT116cas9 cells that are expressing mCherry and citrine due to integration of miReporter-PGK (no. 82477; Addgene).
Nucleic acid delivery. Transfection using Lipofectamine 2000 (no. 11668019; Invitrogen) was performed following the manufacturer's instructions. For nucleofection, cells were nucleofected using Nucleofector SE solution and reagent into a Nucleocuvette following the manufacturer's instructions using the 4D-Nucleofector system (Lonza). The cell number and nucleic acid amounts are indicated in each figure legend. P-EGFP-N1 (plasmid no. 2491; Addgene) was used in transfection and nucleofection experiments as a control.