Journal: mBio

Article Title: Evolutionary dynamics of heparan sulfate utilization by SARS-CoV-2

doi: 10.1128/mbio.01303-25

Figure Lengend Snippet: ACE2-independent binding of the Omicron RBD to cell surface HS. ( A ) CRISPR KO library screening scheme to identify Omicron RBD binding molecules expressed on HEK293T cells. ( B ) Binding of CRISPR KO library-transduced HEK293T cells with (red line) or without (shaded gray) Omicron RBD-Fc, before and after sorting. ( C ) Number of sgRNA sequences identified in Omicron RBD-Fc non-binding cells after sorting. Red: GAG-synthesis-related genes; black: other expressed genes; gray: non-expressed genes in HEK293T cells. ( D ) HS biosynthetic pathway highlighting SLC35B2 and B3GAT3. PAPS, 3'-phosphoadenosine-5'-phosphosulfate; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid; IdoA, iduronic acid. ( E ) Binding of BA.1 RBD-Fc, anti-HS Ab, or anti-CS Ab to mock (black line), or SLC35B2 or B3GAT3 KO (red line) HEK293T cells. ( F ) Binding of WT or BA.1 RBD-Fc, PILRα-Fc, anti-HS Ab, or anti-CS Ab to ACE2 KO HEK293T cells pretreated with (+) or without (–) heparinase or chondroitinase. ( G ) Binding of WT or BA.1 RBD-Fc to HEK293T cells at different heparin concentrations. RBD-Fc binding was normalized to binding in the absence of heparin. ( H ) Immunofluorescence of human nasal tissue with anti-HS Ab and 4', 6-diamidino-2-phenylindole (DAPI). Scale bar, 200 µm. ( I ) Binding of WT or BA.1 RBD-Fc, anti-ACE2 Ab, or anti-HS Ab to cell lines. Data are mean ± SEM of three to four technical replicates. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison tests in panel F ; * P < 0.05, ** P < 0.01, and **** P < 0.0001; ns, not significant. Data are representative of two to three independent experiments.

Article Snippet: CRISPR KO library-transduced HEK293T cells were generated by lentivirus-mediated transduction using the Human CRISPR KO Pooled Library (GeCKO v2; Addgene, 1000000048).

Techniques: Binding Assay, CRISPR, Library Screening, Immunofluorescence, Comparison

Journal: Cancer Cell

Article Title: TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma

doi: 10.1016/j.ccell.2021.07.003

Figure Lengend Snippet: CRISPR screens identify TRIM8 as a regulator of EWS/FLI protein stability and a selective dependency in Ewing sarcoma (A and B) Immunoblot and images showing the expression level and localization of EWS/FLI-GFP in the reporter cell line and subpopulations. ∗ Indicates a non-specific band. (C and D) Schematic of flow cytometry-based CRISPR screening pipeline and the gating strategy used in the screen. (E) Scatterplot showing average log2 fold changes in sgRNA abundance in replicates in the GFP high -sorted population. Negative control guides are highlighted in gray. sgRNAs targeting TRIM8 are highlighted in red. Each dot represents an average of log2 fold changes for four independent sgRNAs per gene. (F and G) Scatterplots showing Ewing sarcoma relative dependency on TRIM8 in screens with the Avana (F) and GecKO (G) libraries. The x axis shows the gene’s dependency score in each cell line. The y axis shows the gene’s dependency rank in an individual cell line. (H and I) Comparison of 14 Ewing sarcoma with 724 other cancer cell lines (H) and 11 Ewing sarcoma with 32 other cancer cell lines (I) demonstrates enrichment of TRIM8 dependency in Ewing sarcoma. Each circle represents a single gene. The x axis shows the effect size, which is the mean difference of dependency scores in Ewing sarcoma cell lines compared with other lines screened. Negative effect size indicates that Ewing sarcoma cells are more dependent on that gene compared with other cancer cell lines screened. The y axis shows the significance calculated as –log10(q value) from empirical-Bayes-moderated t statistics with Benjamini-Hochberg correction. (J) A scatterplot showing ranked disease-enriched dependency in the Avana library (n = 738). The x axis shows the t statistics and the y axis shows the significance calculated as –log10(q value) from empirical Bayes-moderated t statistics with Benjamini-Hochberg correction. PubMed hits represent the number of papers retrieved when searched on PubMed.

Article Snippet: The genome-scale CRISPR-Cas9 screens were performed using the Broad Institute's GeCKOv2 and Avana libraries ( ; ).

Techniques: CRISPR, Western Blot, Expressing, Flow Cytometry, Negative Control, Comparison

Journal: Nature Communications

Article Title: Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

doi: 10.1038/s41467-021-25991-9

Figure Lengend Snippet: a The sequence of the lentiviral gRNAHNF4A and its PAM site in blue. Above is the reference sequence of the HNF4A gene near the gRNA target site, as validated by Sanger sequencing in primary human hepatocytes expressing via lentivirus Cas9 and gRNAscr, with the TSS indicated by a black arrow and the reference protein sequence in red. CGs are bolded and underlined. Below is the dominant Sanger sequence profile of a primary human hepatocyte population expressing lentiviral Cas9 and gRNAHNF4A. This mutation and the resulting difference in the amino acid sequence, as well as the reference sequences at this location, are highlighted in yellow. b Two technical replicates each of the Sanger sequencing chromatograms from the primary human hepatocytes expressing dCas9 and gRNAscr (left) or dCas9 and gRNAHNF4A (right) at the targeted HNF4A locus. c Sanger sequencing results of 13 gRNAscr and 12 gRNAHNF4A DNA strands following bisulfite conversion from the cell populations in ( b ), demonstrating both the methylation levels and the variety of mutations induced by Cas9 in gHNF4A-treated cells. d Same as ( c ) except data expanded is expanded to a larger (>300 bp) region, and simplified such that only CpGs are shown, where blue squares indicate unmethylated CpGs, red squares indicate methylated CpGs, and white squares indicate missing information due to Cas9-induced deletions. CpGs are numbered in accordance with ( a ). e Bisulfite-sequencing data from ( d ) (center) as well as five CpGs immediately upstream (left) and seven CpGs immediately downstream (right), displayed as percent DNA methylation over all sequenced DNA strands in primary human hepatocytes expressing Cas9 and either gRNAscr (gray) or gRNAHNF4A (orange) and as mean ± SD as it is summary data from one mutated cell line. Individual dots represent individual strands of DNA from this clonal cell line. f HNF4A expression in primary human hepatocytes expressing Cas9 and either gRNAscr (gray) or gRNAHNF4A (orange) quantified by RT-qPCR and normalized to GAPDH expression, followed by normalization to average expression in gRNAscr cells, with a dashed line at 1 ( n = 6 independent clones, mean ± SD). * indicates statistically significant difference of P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns not significant (Student’s t -test, two-sided, with Holm-Sidak correction if number of tests is greater than 3). Source data are provided as a Source Data file.

Article Snippet: The HNF4A -targeting gRNA is from the genome-scale CRISPR knock-out (GeCKO) v2 library (purchased as lentiviral plasmid from Genscript) and the FMR1-targeting gRNA from the Jaenisch lab was obtained from Addgene (pgRNA-CGG, Addgene #108248).

Techniques: Sequencing, Expressing, Mutagenesis, Methylation, Methylation Sequencing, DNA Methylation Assay, Quantitative RT-PCR, Clone Assay