Journal: eLife

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: 10.7554/eLife.108724

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 screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single 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. The verification assays were biologically replicated twice. ( 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: eLife

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: 10.7554/eLife.108724

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 screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. 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: eLife

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: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) 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 (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). 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 (n=3). 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 (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). 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 (n=3). 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 (n=5). 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 (n=3). 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. All of these functional effects were biologically replicated at least twice. 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, Functional Assay

Journal: eLife

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: 10.7554/eLife.108724

Figure Lengend Snippet: CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

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

Techniques: CRISPR, Knock-Out, In Vitro

Journal: eLife

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: 10.7554/eLife.108724

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 (n=5 for control, n=7 for sgB4galt1). 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 (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. 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

Journal: Nucleic Acids Research

Article Title: Ints7 deficiency activates DNA damage response to elicit resurgence of endogenous retrovirus MERVL and anastasis of embryonic stem cells

doi: 10.1093/nar/gkaf797

Figure Lengend Snippet: The genome-wide screen reveals DNA damage-related pathways in the activation of MERVL . ( A ) Workflow for the genome-wide CRISPR/Cas9 screen. GeCKO, Genome-scale CRISPR-Cas9 Knockout; LTR, long terminal repeat; MOI, multiplicity of infection; FACS, fluorescence-activated cell sorting. ( B ) FACS analysis showing the percentage of MERVL -activated cells after 5 days of GeCKO library treatment. ( C ) Ranking of previously reported MERVL -negative regulators (left) and positive regulators (right). Genes are ranked by RRA score. RRA, robust ranking aggregation. ( D ) Top five Gene Ontology terms with the lowest P -value identified through the GeCKO screen. ( E ) Immunofluorescence analysis in wild-type mESCs depicting MERVL-Gag (red), γH2AX (yellow), and DAPI (gray). Bar: 10 μm. ( F ) Quantification of γH2AX fluorescence intensity in MERVL + and MERVL − cells. **** P -value < 0.0001 by t -test. ( G ) Immunofluorescence analysis demonstrating MERVL-Gag (red) in mESCs treated with DMSO and 1 and 10 μM etoposide. DMSO, dimethylsulfoxide; ETO, etoposide. Bar: 10 μm. ( H ) qPCR analysis of MERVL expression in mESCs treated with DMSO and 1 and 10 μM etoposide. * P -value < 0.05, **** P -value < 0.0001 by one-way ANOVA, error bars represent the SD. ( I ) Western blot analysis of MERVL-Gag and γH2AX in mESCs collected 1 day after 7 Gy of X-ray irradiation or untreated controls.

Article Snippet: The plasmid DNA library was amplified following the protocol provided by Addgene ( http://www.addgene.org/pooled-library/broadgpp-mouse-knockout-brie/ ).

Techniques: Genome Wide, Activation Assay, CRISPR, Knock-Out, Infection, Fluorescence, FACS, Immunofluorescence, Expressing, Western Blot, Irradiation