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Identification of host factors essential for PEDV infection via genome-wide <t>CRISPR/Cas9</t> screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.
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Identification of host factors essential for PEDV infection via genome-wide <t>CRISPR/Cas9</t> screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.
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Identification of host factors essential for PEDV infection via genome-wide <t>CRISPR/Cas9</t> screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.
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Identification of host factors essential for PEDV infection via genome-wide <t>CRISPR/Cas9</t> screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.
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Identification of host factors essential for PEDV infection via genome-wide <t>CRISPR/Cas9</t> screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.
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Identification of host factors essential for PEDV infection via genome-wide CRISPR/Cas9 screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.

Journal: Journal of Virology

Article Title: IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus

doi: 10.1128/jvi.02028-24

Figure Lengend Snippet: Identification of host factors essential for PEDV infection via genome-wide CRISPR/Cas9 screening. ( A ) Overview of the genome-wide CRISPR/Cas9 screening process conducted in Huh7 cells. Cas9-expressing Huh7 cells were transduced with a genome-wide sgRNA lentiviral library and then infected with PEDV at an MOI of 0.01 or 0.1. Surviving cells were harvested, and sgRNAs were amplified via PCR, followed by quantification of their abundance via next-generation sequencing. The sgRNA abundance from the genome-wide CRISPR/Cas9 screen is shown for infections with 0.01 MOI ( B ) or 0.1 MOI ( C ) of PEDV. The x -axis indicates the number of sgRNAs, whereas the y -axis represents the log 10 value of the normalized sgRNA reads. The 10 most enriched sgRNAs are highlighted in blue and red for clarity. ( D ) IFITM3-wild-type (WT) and IFITM3-KO Huh7 cells were inoculated with PEDV SD at an MOI of 0.05 or 0.1. At 12 h post-infection, the cells were fixed and visualized via immunofluorescence staining with a mouse monoclonal antibody targeting the PEDV nucleocapsid ( N ) protein (red). The cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. ( E ) The viral RNA in the supernatants was quantified by quantitative RT-PCR and is presented as the viral RNA copy number per milliliter. The error bars indicate the standard deviations (s.d.) of three biological replicates ( n = 3). ( F ) Infectious PEDV particles in the supernatants of IFITM3-WT and -KO Huh7 cells were assessed via the TCID 50 assay. The error bars indicate the s.d. from three independent experiments. ***, P < 0.001.

Article Snippet: The human GeCKO v2 CRISPR knockout pooled library module B (Addgene #1000000049) containing 58,028 sgRNAs targeting 19,050 genes was a gift from Feng Zhang ( ).

Techniques: Infection, Genome Wide, CRISPR, Expressing, Transduction, Amplification, Next-Generation Sequencing, Immunofluorescence, Staining, Quantitative RT-PCR