Journal: Cell

Article Title: Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

doi: 10.1016/j.cell.2020.10.030

Figure Lengend Snippet: RAB7A Loss Results in a Reduced Cell Surface Expression and an Increased Endosomal Accumulation of ACE2 (A) Representative histograms of flow cytometry analysis to determine cell surface expression of ACE2 on A549 cell lines (A549 wild-type [WT], A549 ACE2 , and ACE2 with Cas9 and non-targeting [NT] or RAB7A-targeting guide RNAs). The dashed line indicates the gate between the ACE2-negative and -positive cells. (B) Fraction of ACE2 + cells (using gating shown in A). ACE2 expression level was normalized across all samples to the A549 ACE2 cells transduced with non-targeting (NT) guides (n = 2–3 guide RNA-transduced lines per gene, error bars are SEM). (C) Representative images of immunofluorescence staining of ACE2 on A549 ACE2 transduced with an NT or a RAB7A -targeting guide. In NT cells, ACE2 localizes at the cell membrane and in the cytoplasm, while in RAB7A -targeted cells, ACE2 shows a distinct pattern of localization to vesicles. (D) Percent of cells with ACE2 accumulation in vesicles in NT and RAB7A-transduced A549 ACE2 cells (n = 2 biological replicates, error bars are SEM). (E) Representative images of immunofluorescence co-stained for ACE2, EEA1, and LysoTracker in A549 ACE2 cells with a CRISPR guide RNA targeting RAB7A . ACE2 shows a distinct colocalization with EEA1 (an early endosome marker) and a less frequent colocalization with LysoTracker (a lysosomal maker). (F) Representative histograms of flow cytometry analysis to determine cell surface expression in Calu-3 cells. The dashed line indicates the gate between the ACE2-negative and -positive cells. (G) Fraction of ACE2 + cells (using gating shown in F). ACE2 expression level was normalized across all samples to the Calu-3 cells transduced with an NT guide (n = 3 biological replicates, error bars are SEM). (H) Representative images of immunofluorescence staining of ACE2 on Caco-2 cells transduced with a NT or a RAB7A-targeting guide. In NT cells, ACE2 localizes at the cell membrane and in the cytoplasm, whereas in RAB7A-targeted cells, ACE2 shows a distinct pattern of localization to vesicles. (I) Mean area of ACE2 foci in Caco-2 cells transduced with a NT or a RAB7A-targeting guide (n = 4 biological replicates, 80–105 cells per replicate were scored, error bars are SEM). Significance testing for (B) and (G) was performed with a one-way ANOVA (B: F = 9.8, p < 10 −4 ; G: Calu-3: F = 378, p < 10 −4 , Caco-2: F = 222, p < 10 −4 ) with false-discovery rate-corrected post hoc tests. Significance testing for (D) and (I) was performed with an unpaired t test. For all panels, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. See also Figure S6 .

Article Snippet: For each gene of interest, 3 guide RNAs were designed using GUIDES software and were subsequently cloned into an all-in-one vector with an optimized Cas9 scaffold (pCC_01, Addgene 139086) containing Cas9, a guide RNA cassette and a puro resistant cassette ( ; ).

Techniques: Expressing, Flow Cytometry, Transduction, Immunofluorescence, Staining, Membrane, CRISPR, Marker

Journal: Cell

Article Title: Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

doi: 10.1016/j.cell.2020.10.030

Figure Lengend Snippet:

Article Snippet: For each gene of interest, 3 guide RNAs were designed using GUIDES software and were subsequently cloned into an all-in-one vector with an optimized Cas9 scaffold (pCC_01, Addgene 139086) containing Cas9, a guide RNA cassette and a puro resistant cassette ( ; ).

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, Staining, Reverse Transcription, CRISPR, Infection, RNA Sequencing, Software