Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) Flow cytometry analysis of Galectin-9 expression in Jurkat, CCRF-CEM, Loucy, and HPB-ALL scrambled (Scr) and Galectin-9 KO T-ALL cell lines. Representative flow plots are shown (top), and the mean fluorescence intensity (MFI) was calculated of duplicate experiments (bottom) and normalized to the average MFI of Jurkat and CCRF-CEM scrambled control cell lines. A t test was used for statistical analysis. * P < 0.05; n.s., not significant. ( B ) Schematic overview of the genome-wide CRISPR screen for Galectin-9 in T-ALL cell line Jurkat. Created in BioRender. B.Y. (2025) https://BioRender.com/n03l034 . ( C ) Scatterplot showing log 2 fold changes of 20% low versus 20% high expressing Jurkat cells in the genome-wide CRISPR screen assessed by MAGeCK. Dot size indicates −log 10 of the mean FDR of both replicates of a one-sided significance test using a negative binomial model by the MAGeCK algorithm . ( D ) Heatmap depicting the log 2 fold change of 20% low versus 20% high Galectin-9 expressing Jurkat cells in the CRISPR genome-wide and validation screen. Only significant hits of the genome-wide screen and nontargeting controls were included and shown. ( E ) Log 2 fold changes of sgRNA abundance of significant hits IRF1 , TFAP4 , and ADD1 and positive control LGALS9 in the genome-wide CRISPR screen (top) and validation CRISPR screen (bottom). ( F ) Pearson correlation of IRF1 , TFAP4 , and ADD1 expression with LGALS9 expression in the TCGA and TARGET cohorts [UCSC Xena platform ( n = 14,726) ]. See table S1 for abbreviations of cancer types and the number of patients. ( G ) Boxplots depicting log2 IRF1 , TFAP4 , and ADD1 expression (normalized DESeq2 counts+1) in primary T-ALL (TARGET cohort, n = 265) split by low, mid, and high LGALS9 expression. An unpaired t test was used for statistical analysis. ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: Flow Cytometry, Expressing, Fluorescence, Control, Genome Wide, CRISPR, Biomarker Discovery, Positive Control

Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) Western blot quantification in single-cell–derived CRISPR KO clones in Jurkat T-ALL cells. Intensities were normalized to GAPDH and scrambled controls. Error bars indicate SEM of duplicates for which Western blots are shown. Data of scrambled 1+2 represent both scrambled controls together. ( B ) Bar plot showing Galectin-9 concentration secreted in cell culture supernatant after 2 days of culture in Jurkat scrambled control, IRF1 KO, and TFAP4 KO cell lines. Error bars indicate SEM of triplicates. ( C ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR compared to scrambled control in Jurkat scrambled, IRF1 KO, and TFAP4 KO cell lines. Error bars indicate SEM of triplicates. ( D and E ) Western blot (WB) quantification (D) and fold change of LGALS9 expression assessed by RT-qPCR (E) of IRF1-TFAP4 dual KO Jurkat cells. Single-cell–derived scrambled or TFAP4 KO cell lines were subjected to CRISPR editing of IRF1 and scrambled gRNAs. A bulk IRF1 KO sample was taken along as well and note order TFAP4 KO2/1 in WB. WB intensities were normalized to GAPDH and scrambled controls. Error bars indicate SEM of duplicates (D) or triplicates (E). Data of scrambled 1+2 represent both scrambled controls together. ( F ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR of Jurkat cells treated with a range of IFN-γ for 48 hours. Expression was normalized to vehicle-treated scrambled control. Error bars indicate SEM of triplicates. ( G ) Heatmap depicting TF activity analysis [RScenic ] in primary pediatric T-ALL patient samples from the TARGET cohort ( n = 265). Samples were equally split in LGALS9 high, mid, and low expressing groups. See table S2 for other regulons in the same hierarchical cluster as IRF1 and TFAP4. An unpaired t test was used for all statistical analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: Western Blot, Derivative Assay, CRISPR, Clone Assay, Concentration Assay, Cell Culture, Control, Expressing, Quantitative RT-PCR, Activity Assay

Journal: Science Advances

Article Title: Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia

doi: 10.1126/sciadv.ads8351

Figure Lengend Snippet: ( A ) H3K27ac enrichment in the LGALS9 locus analyzed by ChIP-seq experiments in T-ALL cell lines. ( B ) Chromatin looping analyzed by H3K27ac HiChIP-seq experiments in Jurkat and CCRF-CEM cells. Only chromatin looping from the LGALS9 promoter is shown, and three enhancers detected in both cell lines are indicated by an arrow. ( C ) H3K27ac, IRF1, and TFAP4 enrichment in the LGALS9 locus analyzed by ChIP-seq experiments in Jurkat and CCRF-CEM cells. ( D ) Heatmap depicting H3K27ac (left), IRF1 (middle), and TFAP4 (right) enrichment on the LGALS9 promoter and three identified enhancers. Faint gray outline indicates that this region is not peak called in the respective sample. ( E ) Bar plot depicting fold change of LGALS9 expression assessed by RT-qPCR compared to scrambled control in single clone–derived CRISPR-edited Jurkat cells upon a 150-bp deletion in enhancer 1, a 1.6-kb deletion in enhancer 2, or scrambled controls. Error bars indicate SEM of triplicates, and an unpaired t test was used for statistical analysis; * P < 0.05; ** P < 0.01; n.s., not significant. ( F ) Schematic summary of the transcriptional regulation of LGALS9 expression. Created in BioRender. B.Y. (2025) https://BioRender.com/a56h547 .

Article Snippet: Human lentiviral CRISPR library was a gift from X. S. Liu (Addgene no. 1000000132).

Techniques: ChIP-sequencing, HiChIP, Expressing, Quantitative RT-PCR, Control, Derivative Assay, CRISPR

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Stable Transfection, Expressing, Transduction, CRISPR, Amplification

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: The volcano plot shows gene expression changes between cells treated with lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [ ,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Gene Expression, Transduction, CRISPR