mouse crispr knockout pooled library  (Addgene inc)

Journal: Nature Communications

Article Title: Rapid acceleration of KRAS- mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ

doi: 10.1038/s41467-022-30392-7

Figure Lengend Snippet: a – c Pancreatic tissues from KC or KC/Pd mice fed the HFD or Ctrl diet; and KC/Pd mice fed the GW or Ctrl diet for 3 days ( n = 3–4 biologically independent samples). Venn diagram of differentially expressed chemokines ( a ). Quantitative results of Ccl2 protein levels for KC/Pd mice on the GW diet ( b ) and KC or KC/Pd mice on the HFD ( c ). d Representative images of Ccl2 RNAscope in situ hybridization for normal pancreatic tissues of KC/Pd mice fed the GW or Ctrl diet for 3 days ( n = 3 biologically independent samples). e Venn diagram of differentially expressed chemokines for mouse tdTomato RFP–sorted pancreatic epithelial cells of KC/tdPd mice on GW or control diet for 3 days and for the cell culture media from mouse NB490 KPC cells with WT or Ppard KO. f , g Quantitative results of Ccl2 protein levels for tdTomato-RFP + cells ( f , n = 4 biologically independent samples) and for the cell culture media from mouse NB490 KPC cells from 4 independent experiments ( g ). h , i Ccl2 mRNA expression levels in tdTomato-RFP + pancreatic epithelial cells ( h , n = 4 biologically independent samples) and in mouse NB490 KPC cells from 4 independent experiments ( i ). j Ccl2 mRNA expression levels in mouse KC and KC/Pd PDAC cells with or without GW treatment from 4 independent experiments. k , l PPARδ ( k ) and CCL2 ( l ) mRNA expression levels in human PDAC cells transfected with PPARδ siRNAs (siPPARD) or control siRNA (Ctrl) from 3 independent experiments. m The PPARδ binding to the four predicted PPARδ binding sites (pPDBS) in the mCcl2 promoter in mouse KC PDAC cells stably transduced with mouse DDK-tagged PPARδ expressing lentivirus and treated with 1 µM GW or solvent (DMSO) from 3 independent experiments. Data are mean ± SEM. For ( b ), ( f ), ( h ), unpaired two-tailed Student’s t test, for ( c ), ( k – m ), multiple t -tests, for ( g ), ( i ), one-way ANOVA with Bonferroni correction, and for ( j ), two-way ANOVA with Bonferroni correction. * P < .05, ** P < .01, *** P < .001, and **** P < .0001. Source data are provided as a Source Data file.

Article Snippet: We first constructed a mouse Ppard –targeting CRISPR plasmid by subcloning the following pairs of DNA oligos: 5ʹ-(phos) caccgGAGGAAGTGGCCATGGGTGA-3ʹ (sense) and 5ʹ-(phos) aaacTCACCCATGGCCACTTCCTCc-3ʹ (antisense) into pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene, #48139) between two BbsI restriction enzyme sites.

Techniques: RNAscope, In Situ Hybridization, Control, Cell Culture, Expressing, Transfection, Binding Assay, Stable Transfection, Transduction, Solvent, Two Tailed Test

Journal: Nature Communications

Article Title: Rapid acceleration of KRAS- mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ

doi: 10.1038/s41467-022-30392-7

Figure Lengend Snippet: a Representative image of co-staining for Ccl2 RNAscope in situ hybridization with Ccr2 IF (top) or with F4/80 IHC (bottom) in pancreatic normal, acinar-to-ductal metaplasia (ADM), and PanIN tissues in the KC/Pd mice fed the GW diet for 3 days ( n = 5 per group). b KC/Pd mice at 6–8 weeks on the GW diet (50 mg/kg) for 0, 3, or 9 days were euthanized, and then pancreatic tissues were harvested for further analyses ( n = 4–6 per group). Representative images of co-IF staining of Ccr2 with F4/80 (TAMs) (top) or with Gr1 (MDSCs) (bottom). c – h Ccr2 inhibitor PF4136309 (PF) or control solvent (corn oil) was administered via subcutaneous injection at 80 mg/kg twice daily to the KC/Pd mice for 4 days ( n = 5 mice per group), and the mice were fed the GW diet (50 mg/kg) for the last 3 days, and then pancreatic tissues were harvested for further analyses. c Timeline for the mice with PF4136309 and GW diet treatment. d , e Representative images of H&E staining of pancreata ( d ) and percentage of pancreatic neoplastic area per mouse ( e ) for GW-fed KC/Pd mice treated with PF4136309 or Ctrl. f – h Representative images of co-IF staining of Ccr2 with F4/80 (TAMs) ( f , top) or with Gr1 (MDSCs) ( f , bottom), and quantitative co-IF staining results of double-positive cells per 40× field for Ccr2 + /F4/80 + cells ( g ) and Ccr2 + /Gr1 + cells ( h ) for the indicated mice. i Schematic flow showing PPARδ hyperactivation by either the HFD or the GW diet upregulates CCL2, which chemoattracts macrophages and MDSCs into pancreata via the CCL2/CCR2 axis, leading to an inflammatory and immunosuppressive TME (e.g., IL6/STAT3) and subsequent progression of KRAS mu -initiated pancreatic tumorigenesis to PDAC, while PPARD -genetic KO and a CCR2 inhibitor (PF4136309) suppress those tumorigenic effects. Data are mean ± SEM. Unpaired two-tailed Student’s t test. ** P < .01, *** P < .001, and **** P < .0001. Source data are provided as a Source Data file.

Article Snippet: We first constructed a mouse Ppard –targeting CRISPR plasmid by subcloning the following pairs of DNA oligos: 5ʹ-(phos) caccgGAGGAAGTGGCCATGGGTGA-3ʹ (sense) and 5ʹ-(phos) aaacTCACCCATGGCCACTTCCTCc-3ʹ (antisense) into pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene, #48139) between two BbsI restriction enzyme sites.

Techniques: Staining, RNAscope, In Situ Hybridization, Control, Solvent, Injection, Two Tailed Test

Journal: Nature cell biology

Article Title: LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

doi: 10.1038/s41556-021-00672-3

Figure Lengend Snippet: a . A375 shFluc or shLIMIT cells were treated with IFNγ for 24 hours. RNA levels of LIMIT were determined by qRT-PCR. P value by 2-sided t-test. b . A375 shFluc or shLIMIT cells were treated with IFNγ for the indicated time. Protein levels of phospho-STAT1 (p-Y701), STAT1, and GAPDH were determined by Western blotting. 1 of 2 experiments is shown. c . A375 shFluc or shLIMIT cells were treated with IFNγ for 48 hours. Surface expression of HLA-ABC were determined by flow cytometry (FACS). P value by 2-sided t-test. d-g . YUMM1.7 ( d, e ) or CT26 ( f, g ) cells carrying shFluc or shLimit were treated with IFNγ. RNA levels of Limit were determined 24 hours after treatment ( d, f ). Surface staining of MHC-I (H2-D b ) was detected 48 hours after treatment ( e, g ). P value by 2-sided t-test. h-i . A375 WT or LIMIT promoter deletion cells were treated with IFNγ. RNA levels of LIMIT were determined 24 hours after treatment ( h ). Surface expression of HLA-ABC were determined 48 hours after treatment ( i ). P value by 2-sided t-test. j-k . B16 cells were transfected with dCas9-VPR, together with non-targeting sgRNA (sgNT) or sgRNA targeting the promoter of Limit (sgLimit). RNA levels of Limit were determined 24 hours after treatment ( j ). Surface expression of MHC-I (H2-D b ) or PD-L1 were determined 48 hours after treatment ( k ). P value by 2-sided t-test. l . B16-OVA cells carrying shFluc or shB2m were manipulated with Limit CRISPRa (sgLimit) for 48 hours. Surface expression of OVA-H2K b were determined by FACS. P value by 2-sided t-test. m-n . B16-OVA cells were manipulated with B2m knocking down (shB2m) or Limit CRISPRa (sgLimit), and co-cultured with OT-I cell at the ratio of 1:4. Cell death was measured by PI staining in CD45 − tumor cells. Dot plots ( m ) and statistical results ( n ) are shown. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in ( a, c, d, e, f, g, h, i, j, k, l, n ). Source data are provided in Soure_data_Fig2.xlsx and Unmodified_blots_Fig2.pdf.

Article Snippet: To apply CRISPR activation system to activate mouse Limit, phU6-sgRNAs were transfected together with SP-dCas9-VPR (Addgene #63798) into B16 cells.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry, Staining, Transfection, Cell Culture

Journal: Nature cell biology

Article Title: LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

doi: 10.1038/s41556-021-00672-3

Figure Lengend Snippet: a . Co-IP of GBP1-5 with HSP90 antibody in IFNγ-pretreated A375 cells. 1 of 3 experiments is shown. b . Co-IP of HSP90 with Flag antibody in Flag-GBP1-overexpressed A375 cells. * indicated the band shift of HSP90 upon GBP1 overexpression. 1 of 2 experiments is shown. c . Immunofluorescence staining of GBP1 and HSP90 in IFNγ-pretreated A375 cells. 1 of 4 images is shown. d . 293T cells were forced expression of Flag-HSF1 and increased doses of Flag-GBP1. Co-IP of HSF1 or GBP1 with HSP90 antibody were performed 24 hours afterwards. 1 of 2 experiments is shown. e . A375 cells were treated with HSP90 inhibitor, or forced expression of GBP1. Indicated proteins were detected 48 hours afterwards. 1 of 2 experiments is shown. f-g . A375 cells were treated with IFNγ and KRIBB11. RNA ( f ) or surface staining ( g ) levels of indicated genes were determined 48 hours afterwards. P value by 2-sided t-test. h . YUMM1.7 shFluc or shHsf1 cells were treated with IFNγ. Total protein ( h ) or surface expression ( i ) levels of indicated genes were determined 48 hours afterwards. 1 of 2 experiments is shown. P value by 2-sided t-test. j-k . Tumor growth curves of YUMM1.7 shFluc or shHsf1 cells in NSG mice ( j ) or wild type C57BL/6 mice ( k ). n = 5 ( j ) or 6 ( k ) animals, P value by 2-sided t-test for end point tumor volume. l . Percentages of CD3 + , Ki67 + , IFNγ + , and TNFα + T cells in YUMM1.7 shFluc or shHsf1 tumors. n = 5 biological independent samples, P value by 2-sided t-test. m . A375 shFluc or shLIMIT cells were transfected with HSE-luc and PRL-SV40 overnight, and then treated with IFNγ for additional 48 hours. HSF1 transcriptional activity is depicted as the relative luciferase activity. P value by 2-sided t-test. n . B16 cells were manipulated with Limit CRISPRa and treated with KRIBB11. Surface expression of MHC-I (H2-D b ) was determined 48 hours afterwards. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in ( f, g, i, m, n ). Source data are provided in Soure_data_Fig6.xlsx and Unmodified_blots_Fig6.pdf.

Article Snippet: To apply CRISPR activation system to activate mouse Limit, phU6-sgRNAs were transfected together with SP-dCas9-VPR (Addgene #63798) into B16 cells.

Techniques: Co-Immunoprecipitation Assay, Electrophoretic Mobility Shift Assay, Over Expression, Immunofluorescence, Staining, Expressing, Transfection, Activity Assay, Luciferase

Journal: Nucleic Acids Research

Article Title: Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

doi: 10.1093/nar/gkx1222

Figure Lengend Snippet: Design, optimization and cross-comparison of HIT transcription activation systems. ( A–C ) Cartoons illuminating the mechanisms of optimized 4-OHT induced transcription activation using a direct fusion HIT construct (A), the HIT-SAM system (B), and the HIT-SunTag system (C). ( D–I ) Cross-comparison among three optimized HIT transcription activation systems. Transcription activation induced by HIT constructs was examined in the luciferase reporter assay (D), by quantification of relative mRNA level of endogenous expression for Klf4 (E) and Oct4 (F), and by flow-cytometry analyses of CD43 protein level on the cell surface (G–I). Representative plots (G) and quantitative analyses of median CD43 fluorescent intensities (H) and overall CD43 fluorescent intensities (I) of CD43+GFP+ positive population were shown. GFP fluorescence indicates successful transfection. Cells transfected with the same amount of reporter construct or sgRNA only while keeping the total amount of transfection constant were used as negative controls (NC) in the luciferase reporter assay. Cells transfected with an ER T2 tagged GFP construct while keeping the total amount of transfection constant were used as negative control (NC) in qRT-PCR assays. Cells transfected with an unrelated sgRNA (ctl sgRNA) and GFP were used as negative control (NC) in flow-cytometry analyses. Data showed mean ± SD. n = 3 biological replicates. ns: non-significant;* P < 0.05; ** P < 0.01; *** P < 0.001; two tailed t -tests. Three biological replicates means three independently transfected samples throughout this study. The readouts without drug induction were compared in t-tests against the negative controls (NCs) for background detection.

Article Snippet: One sgRNA displaying highest activity was chosen for each gene ( ). sgRNA2.0s for the SAM system were cloned using a backbone plasmid from Feng Zhang (Addgene plasmid # 61424) ( ). sgRNAs candidates of SaCas9 for both gene editing and activation were chosen by PAM sequence requirement (NNGRRT) ( ) ( ).

Techniques: Comparison, Activation Assay, Construct, Luciferase, Reporter Assay, Expressing, Flow Cytometry, Fluorescence, Transfection, Negative Control, Quantitative RT-PCR, Two Tailed Test