Journal: The Journal of Biological Chemistry

Article Title: The RNA methyltransferase NSUN2 catalyzes 5-methylcytosine (m 5 C) on IL1B mRNA to promote transcript stability

doi: 10.1016/j.jbc.2026.111290

Figure Lengend Snippet: Nsun2 reduction decelerates the advancement of systemic inflammation in mice. A , the m 5 C methylation level in total RNA of mouse lung tissues administrated with LPS was determined by m 5 C dot blot assay. Methylene blue (MB) staining was set as an internal reference for sample loading. The histogram summarized the ratios of the dot intensities of m 5 C versus methylene blue . Data are means ± SD, n = 6. B and C , published RNA-seq data derived from other researchers was downloaded from the GEO database. We reanalyzed the expression profiles of m 5 C modification-associated genes in lung tissues from mice with systemic inflammation. These systemic inflammation mouse models were conducted by three common methods: LPS treatment ( GSE217695 ), cecal microbiome (CM) treatment ( GSE239388 ), as well as cecal ligation and puncture treatment (CLP) ( GSE179554 ). B , heatmap represents normalized gene expression ( red , high expression; blue , low expression). C , the histogram summarized the relative expression of Nsun2 . Data are means ± SD, n = 3 or 4. D , schema for the strategy used to generate the Nsun2 knockout mice via the CRISPR/Cas9 technology. E , PCR identification of genotypes of C57BL/6N wild-type (WT) mice and Nsun2 mutant (heterozygous) mice. F , determine the protein expression of NSUN2 in lung tissues of WT or Nsun2 +/− mice by IHC. The scale bar represents 100 and 50 μm (high magnification). G , timeline for LPS-induced systemic inflammation as well as body weight measurements and behavioral assessment (15 mice per group). H , weight changes of Nsun2 +/− and control WT mice before injection (0 h) and 16, 18, 20, 22, and 24 h after LPS or PBS injection. The significance of the weight change differences between WT + LPS group and Nsun2 +/− + LPS group was analyzed and plot on the weight change polyline of Nsun2 +/− + LPS group. Weight change (%) = (weight at a specific time-weight at 0 h)/weight at 0 h × 100% (15 mice per group). I , the statistical analysis of the M-CASS scores was measured at 24 h after LPS administration (n = 15). J , representative H&E staining of mouse lung sections from each group. The degree of lung damage was measured via the lung injury scoring system (n = 15). The scale bar represents 100 and 50 μm (high magnification). K , quantitative real-time PCR analysis of the mRNA levels of Il1b , Il6 , Cxcl10 , and Ccl2 in lung tissues. Gapdh served as a loading control. Data are means ± SD, n = 15. Trdmt1 , tRNA aspartic acid methyltransferase 1; Tet , tet methylcytosine dioxygenase; Alkbh , alkB homolog 1. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. CCL2, C-C motif chemokine ligand 2; CXCL10, C-X-C motif chemokine ligand 10; GEO, Gene Expression Omnibus; IHC, immunohistochemical; IL1B, interleukin 1 beta; LPS, lipopolysaccharides; M-CASS, mouse clinical assessment score for sepsis; m5C, 5-methylcytosine; NSUN, NOP2/Sun RNA methyltransferase.

Article Snippet: Nsun2 +/− mice were derived from C57BL/6N mice employing CRISPR/Cas-mediated genome engineering scheme (Mice Nsun2 Gene ID: 28114) in Cyagen Biotechnology Co, Ltd The wild-type and Nsun2 gene trap alleles were distinguished by polymerase chain reaction (PCR) with One Step Mouse Genotyping Kit (Vazyme).

Techniques: Methylation, Dot Blot, Staining, RNA Sequencing, Derivative Assay, Expressing, Modification, Ligation, Gene Expression, Knock-Out, CRISPR, Mutagenesis, Control, Injection, Real-time Polymerase Chain Reaction, Immunohistochemical staining

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: bioRxiv

Article Title: PARG inhibition sequesters nuclear PAR-binding proteins, including XRCC1 and its partners, into nuclear condensates to elicit cytotoxicity

doi: 10.64898/2026.03.18.712393

Figure Lengend Snippet: A. V-abl B cells expressing Flag-tagged Cas9 were infected with a BFP-expressing lentiviral gRNA library. BFP+ cells were sorted and treated with doxycycline for five days to induce Cas9 expression. Indicated inhibitors treatment was performed at IC90 for six days. Then, DNA was collected from the cells for sequencing and analysis using the MAGeCK pipeline. B. Scatter plot of total CRISPR genes Z-scores for Niraparib and Olaparib IC90 treatments. Simple linear regression and Pearson correlation test were performed to determine R. C. Scatter plot of total CRISPR genes Z-scores for PDD0017273 and Olaparib IC90 treatments. Simple linear regression and Pearson correlation test were performed to determine R. D. Summary table of Pearson R coefficient for linear regression between cell lines sensitivity to PDD, olaparib, nirabarib and talazoparib. Depmap sensitivity score (PRISM repurposing data) of 536 cell lines to the indicated compound. Simple linear regression and Pearson correlation test were performed to determine R. Colored scaled is represented on the right and R coefficients are written in white. E. Heatmap representing z-score (color range) and False Discovery Rate FDR (size) for the genes targeting indicated in rows, in cells challenged with inhibitor indicated above, in columns. Gene implicated in homologous recombination are displayed. F. CRISPR screen z-scores ranking of co-essential genes with PDD00017273 treatment. SSBR related genes are shown in red, ADP-ribosylation related genes are shown in dark blue, HR related genes are shown in light grey.

Article Snippet: 100 million cells were infected with a BFP-expressing gRNA lentiviral library (Addgene Pooled Library #67988).

Techniques: Expressing, Infection, Sequencing, CRISPR, Homologous Recombination