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Journal: Cell reports
Article Title: CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.
doi: 10.1016/j.celrep.2023.112076
Figure Lengend Snippet: Figure 1. Genome-wide CRISPR-Cas9 screen identifies host-encoded regulators of SARS-CoV-2 frameshifting (A) Schematic of the SARS-CoV-2 genome. Dotted box indicates close up of region shown in (B) harboring the coronavirus frameshifting element (FSE). (B) Secondary structure of the SARS-CoV-2 FSE containing the slippery sequence and three-stemmed pseudoknot. Based on structural data from Bhatt et al.13
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data CRISPR screening data This paper GEO: GSE206101 Experimental models: Cell lines HCT116 ATCC CCL-247; RRID: CVCL_0291 HEK293T ATCC CRL-3216; RRID: CVCL_0063 VeroE6 ATCC CRL-1586; RRID: CVCL_0574 HCT116-SARS-CoV-2-PRF-1 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-1 reporter cell line 2 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 2 This paper N/A HCT116-ACE2-Blast cell line 1 This paper N/A HCT116-ACE2-Blast cell line 2 This paper N/A Oligonucleotides Sequences of oligonucleotides used in this study are provided in Table S2 This
Techniques: Genome Wide, CRISPR, Sequencing
Journal: Cell reports
Article Title: CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication.
doi: 10.1016/j.celrep.2023.112076
Figure Lengend Snippet: Figure 6. Loss of ribosome recycling factors inhibits SARS-CoV-2 replication and reduces ribosomal frameshifting during infection (A) Experimental workflow for testing the effect of ribosome recycling on SARS-CoV-2 replication. (1) Lentiviral expression of ACE2 in HCT116 cells. (2) CRISPR- Cas9-mediated knockout of ABCE1 or DENR. (3) Infection with SARS-CoV-2. (4) Sample collection 7 h post-infection and qRT-PCR analysis of nucleocapsid (N) expression. (B) Immunoblotting of ABCE1 and DENR in HCT116-ACE2 CRISPR knockout pools. (C and D) qRT-PCR measurement of nucleocapsid mRNA expression 7 h after SARS-CoV-2 infection in cells transduced with non-target control sgRNA (sgNeg) or sgRNAs targeting ABCE1 (C) or DENR (D). Two distinct sgRNAs were used per gene in two independent ACE2-expressing HCT116 cell lines (ACE2-1 and ACE2-2). Nucleocapsid expression was normalized to host GAPDH expression. (E) Schematic of SARS-CoV-2 ORF1a and ORF1b non-structural proteins (NSPs). Antibody symbols indicate upstream (NSP1) and downstream (NSP16) NSPs that were detected by immunoblotting to assess relative frameshifting rate. (F) Representative western blot for NSP1, NSP16, and nucleocapsid from uninfected cells, infected control cells (sgNeg) and infected ABCE1 knockout pools generated with two independent sgRNAs (sgABCE1-1 and sgABCE1-2). (G) Quantification of NSP1, NSP16, and nucleocapsid protein levels, normalized to host GAPDH expression, from three independent experiments. Data are represented as the mean ± SD with individual replicates plotted. The p values for qRT-PCR experiments were calculated by two-way ANOVA with Dunnett’s multiple comparisons test. The p values for the immunoblotting results were calculated by two-way ANOVA with Tukey’s multiple comparisons test; **p % 0.01, ***p % 0.001; n = 3 biological replicates for all experiments.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data CRISPR screening data This paper GEO: GSE206101 Experimental models: Cell lines HCT116 ATCC CCL-247; RRID: CVCL_0291 HEK293T ATCC CRL-3216; RRID: CVCL_0063 VeroE6 ATCC CRL-1586; RRID: CVCL_0574 HCT116-SARS-CoV-2-PRF-1 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-1 reporter cell line 2 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 1 This paper N/A HCT116-SARS-CoV-2-PRF-0 reporter cell line 2 This paper N/A HCT116-ACE2-Blast cell line 1 This paper N/A HCT116-ACE2-Blast cell line 2 This paper N/A Oligonucleotides Sequences of oligonucleotides used in this study are provided in Table S2 This
Techniques: Infection, Expressing, CRISPR, Knock-Out, Quantitative RT-PCR, Western Blot, Transduction, Control, Generated