crispr library designer  (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