Journal: Cell Reports Medicine

Article Title: Targeting the mevalonate pathway potentiates NUAK1 inhibition-induced immunogenic cell death and antitumor immunity

doi: 10.1016/j.xcrm.2024.101913

Figure Lengend Snippet: NUAK1 inhibition elicits immunogenic death of tumor cells (A) Workflow of CRISPR screen for identifying ICD regulators. (B) Rank plot of the top enriched genes in CRISPR knockout screen processed by MAGCK. (C) Boxplot of NUAK1 expression in tumor tissues compared to normal tissues in patients with colorectal adenocarcinoma (COAD). (D) FACS analysis of ecto-CALR in Nuak1-deficient MC38 and AKR cell lines ( n = 3). (E) The mean fluorescence intensity (MFI) of MC38 and AKR cell lines treated by vehicle and 10 μM or 20 μM HTH-01-015 for 24 h ( n = 3). (F) Apoptosis in MC38 and AKR cell lines upon vehicle and 10 μM or 20 μM HTH-01-015 treatment for 24 h ( n = 3). (G) The immunofluorescence staining of CALR in MC38 and AKR cells treated with vehicle or 20 μM HTH-01-015 for 24 h. Scale bar, 1 μm. (H) The extracellular ATP in vehicle or 20 μM HTH-01-015-treated MC38 and AKR cell cultures ( n = 4). (I) HMGB1 in MC38 and AKR cell lines was detected by immunofluorescence staining treated with vehicle or 20 μM HTH-01-015 for 24 h. Scale bar, 2 μm. (J) Western blots of secreted HMGB1 in cell culture supernatant treated with vehicle or 20 μM HTH-01-015 for 24 h and total protein bands were visualized by UV imaging, and the quantification was performed using ImageJ. (K) Phagocytosis of antigens from vehicle and 10 μM or 20 μM HTH-01-015-treated MC38 tumor cells (CFSE-labeled) by BMDCs or macrophages co-cultured for 12 h ( n = 4). (L) FACS analysis of the activation marker CD107a and proliferation marker Ki67 of OT-1 cells priming by antigen pre-loading BMDCs that co-cultured with vehicle or 20 μM HTH-01-015-treated OVA antigen expression MC38 cells lines for 12 h ( n = 4). Data are presented as the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, by Student’s t test (D, H, and L) or one-way ANOVA (E and K).

Article Snippet: Mouse Kinome CRISPR Knockout Library (Brie) , Addgene , Cat# 75316.

Techniques: Inhibition, CRISPR, Knock-Out, Expressing, Fluorescence, Immunofluorescence, Staining, Western Blot, Cell Culture, Imaging, Labeling, Activation Assay, Marker

Journal: Cell Reports Medicine

Article Title: Targeting the mevalonate pathway potentiates NUAK1 inhibition-induced immunogenic cell death and antitumor immunity

doi: 10.1016/j.xcrm.2024.101913

Figure Lengend Snippet:

Article Snippet: Mouse Kinome CRISPR Knockout Library (Brie) , Addgene , Cat# 75316.

Techniques: Control, Recombinant, ATP Assay, Protein Extraction, Activation Assay, Amplex Red Cholesterol Assay, Bicinchoninic Acid Protein Assay, CRISPR, Knock-Out, Software

Journal: bioRxiv

Article Title: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC reprogramming

doi: 10.1101/2023.07.31.551297

Figure Lengend Snippet: ( A ) Experimental design. X-GFP iCas9 ESCs were infected with a genome-wide lentiviral gRNA library and differentiated into NPCs. Next, NPCs were treated with doxycycline to activate the expression of the reprogramming cassette and the iCas9 . The knockouts took place during reprogramming. At day 10 of reprogramming, three populations were FACS-separated: non-pluripotent (SSEA1-X-GFP-), early pluripotent (SSEA1+ X-GFP-) and late pluripotent, X-reactivated (SSEA1+ X-GFP+). For these three populations and the NPCs, genomic DNA extraction, PCR-amplification of the gRNA sequences, sequencing and analysis of gRNA abundance were performed. n=2 biological replicates (independent reprogramming rounds). ( B ) Pathways related to overrepresented genes in the three reprogramming populations (non-pluripotent, early pluripotent and late pluripotent) compared to NPCs (WikiPathways Mouse 2019). For pathway enrichment analysis, the top 250 genes from each individual comparison to NPCs ranked by RRA score with MAGeCK software were merged and selected. ( C ) gRNA abundance comparison (early pluripotent vs non-pluripotent) and representation of genes with negative Log2FC (underrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = −0.75) (activators of early pluripotency). ( D ) gRNA abundance comparison (late pluripotent vs early pluripotent) and representation of genes with negative Log2FC (underrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = −0.75) (activators of late pluripotency, X-reactivation). Genes highlighted in yellow are related to pluripotency, genes highlighted in red are involved in Notch or interferon γ signaling. ( E ) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in “late pluripotent vs early pluripotent” comparison (activators of late pluripotency, X-reactivation, n=1313 genes). Pathways related to proliferation, differentiation and metabolism are shown in gray. The rest of the pathways are highlighted in green. RRA score < 0.05 and Log2FC < −0.8 filtering was applied. ( F ) Experimental design for (G). Treatment with molecules targeting pathways identified in the CRISPR screen was done at the beginning of reprogramming (day 0 - day 5), later in reprogramming (day 5 - day 7) or during the whole process (day 0 - day 7). At day 7, flow cytometry analysis of SSEA1 and X-GFP percentages was performed. ( G ) Pathway validation by molecule treatment at the beginning of reprogramming (day 0 - day 5), later in reprogramming (day 5 - day 7) or during the whole process (day 0 - day 7) in three independent reprogramming rounds, n=3 (except for TGFβ, n=2). Flow cytometry analysis at day 7 of SSEA1 and X-GFP percentages is shown. Data represented as mean +/− SD. Statistics (paired t-tests): where not specified = non-significant; * = p<0.05; ** = p<0.01; *** = p<0.001.

Article Snippet: The gRNA library used for the screening was the Mouse Improved Genome-wide Knockout CRISPR Library v2 (a gift from Kosuke Yusa, Addgene, #67988) , with 90,230 gRNAs targeting 18,424 genes (average of 5 gRNAs per gene).

Techniques: Infection, Genome Wide, Expressing, DNA Extraction, Amplification, Sequencing, Comparison, Software, CRISPR, Flow Cytometry, Biomarker Discovery

Journal: bioRxiv

Article Title: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC reprogramming

doi: 10.1101/2023.07.31.551297

Figure Lengend Snippet: A genome-wide CRISPR knockout screen reveals molecular networks involved in reprogramming and X-chromosome reactivation. Related to . (A) Validation of knockout efficiency by flow cytometry. Flow cytometry analysis during 6 days of doxycycline treatment in the X-GFP iCas9 ESC line was done to measure the X-GFP percentage decay in cells containing a gRNA targeting the GFP gene. Gating shows the X-GFP+ population. (B) Percentage of gRNA representation in the plasmid library, infected ESCs and the 4 populations analyzed in two independent screening rounds: NPCs and day 10 reprogramming populations (non-pluripotent, early pluripotent, late pluripotent). Error bars represent SD. ( C ) gRNA abundance comparisons (related to D-G): NPCs to non-pluripotent, early pluripotent and late pluripotent populations. ( D ) Pathways related to common underrepresented genes (n=927 genes) in the three reprogramming populations compared to NPCs (WikiPathways Mouse 2019). For all comparisons, a RRA score < 0.05 and Log2FC < −0.75 (underrepresented) filtering was applied. ( E-G ) Representation of genes with negative Log2FC (underrepresented) vs −log10 RRA in the non-pluripotent (E), early pluripotent (F) and late pluripotent (G) populations compared to NPCs (RRA cutoff = 0.05, Log2FC cutoff = −0.75). ( H ) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in the “early pluripotent vs non-pluripotent” comparison (activators of early pluripotency, n=1361 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied). ( I ) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the “early pluripotent vs non-pluripotent” comparison (repressors of early pluripotency, n=693 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied). ( J ) Representation of genes with positive Log2FC (overrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the “early pluripotent vs non-pluripotent” comparison (repressors of early pluripotency). ( K ) Representation of genes with positive Log2FC (overrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the “late pluripotent vs early pluripotent” comparison (repressors of late pluripotency, X-reactivation). ( L ) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the “late pluripotent vs early pluripotent” comparison (repressors of late pluripotency, X-reactivation, n=839 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied).

Article Snippet: The gRNA library used for the screening was the Mouse Improved Genome-wide Knockout CRISPR Library v2 (a gift from Kosuke Yusa, Addgene, #67988) , with 90,230 gRNAs targeting 18,424 genes (average of 5 gRNAs per gene).

Techniques: Genome Wide, CRISPR, Knock-Out, Biomarker Discovery, Flow Cytometry, Plasmid Preparation, Infection, Comparison

Journal: bioRxiv

Article Title: Catalytic-independent functions of INTAC in conferring sensitivity to BET inhibition

doi: 10.1101/2024.02.07.579305

Figure Lengend Snippet: (A) Flow chart of the genome-wide CRISPR screening conducted to enrich cells with acquired resistance to BET inhibition in Eμ-Myc cells with the stable expression of Cas9. (B) Rank plot showing the log2 fold change (JQ1 versus DMSO) of sgRNA reads in survived cells post JQ1 treatment. Top ten enriched sgRNA targets and genes encoding INTAC complex labeled in color. (C) Schematic of the INTAC complex, components of the auxiliary module are highlighted. (D) Western blotting for INTS10, INTS13, INTS14 and INTS15 in CRISPR knockout Eμ-Myc cells. β-actin is a loading control. (E) Cell survival assays in sgCtr, sgINTS10, sgINTS13, sgINTS14 and sgINTS15 Eμ-Myc cells treated with DMSO or JQ1. (F) Western blotting for INTS1, INTS8 and INTS11 in CRISPR knockout Eμ-Myc cells. (G) Cell survival assays in sgCtr, sgINTS1, sgINTS8 and sgINTS11 Eμ-Myc cells treated with DMSO or JQ1. (H) Measurement for transcription termination of the representative snRNAs Rnu1a1 and Rnu3b2 in sgCtr, sgINTS15, sgINTS10, sgINTS8 and sgINTS11 Eμ-Myc cells. (I) Western blotting for RNA Pol II phosphorylation levels at carboxyl-terminal domain (CTD) Serine 5 (pSer5) and Serine 2 (pSer2) in sgCtr, sgINTS15, sgINTS10, sgINTS8 and sgINTS11 Eμ-Myc cells. β-actin is a loading control. See also Figure S1.

Article Snippet: Mouse Two Plasmid Activity-Optimized CRISPR Knockout Library (Addgene#1000000096) was amplified in E coli.

Techniques: Genome Wide, CRISPR, Inhibition, Expressing, Labeling, Western Blot, Knock-Out, Control, Phospho-proteomics

Journal: Molecular Therapy Oncolytics

Article Title: Advancements in CRISPR screens for the development of cancer immunotherapy strategies

doi: 10.1016/j.omto.2023.100733

Figure Lengend Snippet: CRISPR screens in T cells to develop advanced T-cell-based products for cancer immunotherapy

Article Snippet: Mouse , CD4 + T cells , loss of function , library pMSCV-U6gRNA(lib)-PGKpuroT2ABFP (Addgene: #104861) , retrovirus transduction , expression of IRF4, XBP1, or GATA3 , Pparg and Bhlhe40 , PPARG (peroxisome proliferator activated receptor gamma) and BHLHE40 (basic-helix-loop-helix protein 40) , PPARG and BHLHE40 are crucial to TH2 gene regulation and differentiation. Genes regulating TH2 activation and genes regulating TH2 differentiation are highly overlapped. , Henriksson et al. (2019) .

Techniques: CRISPR, Transduction, Selection, Expressing, Activation Assay, Retroviral, Genome Wide, Knock-Out, In Vitro, Activity Assay, Electroporation, Membrane, Adoptive Transfer Assay, Transgenic Assay, Infection, In Vivo, Migration, Biomarker Discovery, Immunofluorescence, Staining, Flow Cytometry