Journal: bioRxiv

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.1101/2025.08.20.671236

Figure Lengend Snippet: a. Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. b. Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. c. Verification of candidate genes by individual gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. d. GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: Ex Vivo, CRISPR, Genome Wide, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.1101/2025.08.20.671236

Figure Lengend Snippet: a. Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. b. Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The p-values were calculated using the α-RRA algorithm in MAGeCK. c. Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The p-values were calculated using the α-RRA algorithm in MAGeCK.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: In Vivo, CRISPR, Ex Vivo

Journal: bioRxiv

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.1101/2025.08.20.671236

Figure Lengend Snippet: a. CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS. The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR. The p-values were calculated using a two-tailed Student’s t-test. b. The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1. The p-values were calculated using a two-tailed Student’s t-test. c. CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR. The secreted TNFα and IFNγ in medium were measured by ELISA. The p-values were calculated using a two-tailed Student’s t-test. d. CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells. The p-values were calculated using a two-tailed Student’s t-test. e. Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. f. Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells. The p-values were calculated using a two-tailed Student’s t-test. g. Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA. The p-values were calculated using a two-tailed Student’s t-test. h. Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. i. Volcano plot showing upregulated and downregulated genes (p-value<0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini-Hochberg with the R package DESeq2 (version 1.22.2). j. Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: CRISPR, Knock-Out, Expressing, Co-Culture Assay, Quantitative RT-PCR, Two Tailed Test, Over Expression, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, shRNA, Control, Labeling

Journal: bioRxiv

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.1101/2025.08.20.671236

Figure Lengend Snippet: a. Schematic view of B4galt1 functional test in tumor microenvironment. b. CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. c. Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted. The p-value was calculated using a two-tailed Student’s t-test. d. CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors. The p-value was calculated using a two-tailed Student’s t-test. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: Functional Assay, CRISPR, Knock-Out, Control, In Vivo, Two Tailed Test