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. 2022 Jun 7;34(6):857–873.e9. doi: 10.1016/j.cmet.2022.04.009

Figure 5.

Figure 5

SARS-CoV-2 infections in BSG KO kidney organoids

(A) Schematic of Cas9/gRNA-targeting sites (pink arrows) in BSG locus showing exon structure (blue boxes) and PCR amplicons (light gray boxes in all figures in this study). Histogram shows allelic sequence distribution after the transfection of the different gRNAs in undifferentiated ES[4] cells expressing an inducible Cas9 (iCas9). WT, wild type; mut, mutation; FS, frameshift.

(B) Representative sequence of the wild type (+/+) or BSG mutant clones generated with the different gRNAs.

(C) Representative bright-field images of WT and BSG KO kidney organoids. Scale bars, 250 μm. Hematoxylin and eosin staining show tubular-like (∗∗) and glomerular-like (∗) structures. Scale bars, 250 and 50 μm (magnified views).

(D) mRNA expression levels of BSG in WT and BSG KO kidney organoids by qPCR. Data are mean ± SD. n = 1 independent experiment from a pool of 12 organoids/group with at least two technical replicates each.

(E) Experimental scheme for the infection with SARS-CoV-2 of WT and BSG KO kidney organoids.

(F) TEM analysis of WT and BSG KO kidney organoids infected with SARS-CoV-2 at 3 dpi. Representative images of infected BSG WT specimens (left) show numerous viral particles in the cell surface of a dying cell (1 and 2). Details for podocyte-like cells (3) exhibiting podocyte-related structures including primary (pp) and the deposition of a basement membrane (bm). Scale bars, 2 μm, 200 nm (magnified views in 1 and 2), and 2 μm (magnified views in 3). Representative images of infected BSG KO specimens (right) show numerous viral particles in the intercellular space (4) and in the cell surface (5). Details for podocyte-like cells (6) exhibiting podocyte-related structures including primary (pp) and the deposition of a basement membrane (bm). Scale bars, 2 and 5 μm, 200 nm (magnified views in 4 and 5), and 1 μm (magnified view in 6).

(G) SARS-CoV-2 mRNA expression levels in mock-treated or SARS-CoV-2-infected WT and BSG KO kidney organoids at 3 dpi by qPCR. Data are mean ± SD. n = 1 independent experiment from a pool of 12 organoids/group with at least two technical replicates each.

(H) Immunofluorescence of mock or SARS-CoV-2-infected WT and BSG KO kidney organoids at 3 dpi for ACE2 (green), viral nuclear protein (NP, red), LTL (gray), and DAPI (blue). Scale bars, 250 μm.

See also Figure S8.