Journal: Life Science Alliance

Article Title: In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer

doi: 10.26508/lsa.202000770

Figure Lengend Snippet: (A) PTEN and SPRY2 genomic alterations in metastatic prostate cancer patients with taxane treatment (SU2C/PCF Dream Team, 2015). (B) Non-cystic prostate tumour weights from indicated mice at clinical end point ( Pten −/− , n = 5; Pten −/− Spry2 −/+ , n = 6; * P < 0.05; Mann–Whitney test; mean values ± SD are shown). (C) Kaplan–Meier plot for overall survival of SP1 prostate orthograft bearing mice treated as indicated (log-rank Mantel–Cox test). (D) IHC quantification of Ki67 staining in SP1 prostate tumour orthograft sections from CD-1 nude immunocompromised mice treated as indicated (vehicle, n = 5; docetaxel, n = 4; * P < 0.05; Mann–Whitney test; mean values ± SD are shown). (E) Schematic of the workflow of the CRISPR drop-out screen, bioinformatics analysis and target validation. (F) Western blot images to confirm expression of Cas9 in whole cell lysates from SP1 cells transfected with Cas9-EGFP. α-tubulin is used as a loading control. (G) Western blot images to confirm expression of Cas9 cytoplasmic and nuclear extracts from FACS-sorted SP1 cells with stable Cas9-EGFP expression. Lamin B and α-tubulin were used as nuclear and cytosolic markers, respectively. (H) Schematic illustration of in vivo CRISPR/Cas9 screen. SP1 cells were stably transfected with Cas9-EGFP. After double FACS sorting, SP1 cells with stable expression of Cas9 were selected and amplified for the screen. GeCKO2 V2 whole genome sgRNA library A was used for lentiviral production and transduction of SP1 Cas9-EGFP cells. After 7 d of puromycin selection, the infected SP1 cells were injected in the anterior prostates of CD1-immunocompromised mice. After 7 d of recovery, mice were randomised and treated with vehicle (n = 9) or docetaxel (n = 5). (I) sgRNA transfected SP1 prostate orthograft burden in CD-1 nude immunocompromised mice treated as indicated (Vehicle, n = 9; docetaxel, n = 5; ns, not significant; Mann–Whitney test; mean values ± SD are shown). Source data are available for this figure.

Article Snippet: SP1-Cas9 cells were transduced with the CRISPR library (GeCKOv2 library A; Addgene) and 10 7 cells were injected into one of the anterior prostates of each CD-1 nude mouse.

Techniques: MANN-WHITNEY, Staining, CRISPR, Biomarker Discovery, Western Blot, Expressing, Transfection, Control, In Vivo, Stable Transfection, Amplification, Transduction, Selection, Infection, Injection

Journal: Life Science Alliance

Article Title: In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer

doi: 10.26508/lsa.202000770

Figure Lengend Snippet: Development of an orthograft model for a CRISPR/Cas9 screen. (A) Representative images of a prostate tumour from a Probasin-Cre Pten fl/fl Spry2 fl/+ mouse, SP1 primary cell line established from a prostate tumour, and SP1 prostate orthograft generated by injecting SP1 cells in the anterior prostate of a CD-1 nude immunocompromised mouse. Scale bars = 100 μm. (B) Western blot image of androgen receptor expression in SP1 cells. α-tubulin is used as a housekeeping gene. (C) Representative immunostained images of SP1 prostate orthograft sections from CD-1 nude immunocompromised mice treated as indicated (vehicle, n = 5; docetaxel, n = 4). Scale bar = 100 μm. (D) Detailed workflow of in vivo CRISPR/Cas9 screen.

Article Snippet: SP1-Cas9 cells were transduced with the CRISPR library (GeCKOv2 library A; Addgene) and 10 7 cells were injected into one of the anterior prostates of each CD-1 nude mouse.

Techniques: CRISPR, Generated, Western Blot, Expressing, In Vivo

Journal: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet: ( A ) For genome-wide screen, over 100,000 plasmids, each containing a guide RNA towards different early consecutive exons, were packaged into lentiviral particles. Cas9-expressing ER-Hoxb8 cells were pool-transduced, selected, and differentiated to induce S100A9 expression. Hits and reference cells were collected by sorting according to their phenotypes of interest. DNA of both samples was purified for next-generation sequencing and subsequent analysis. ( B ) Precursor and differentiated Cas9 and Cas9-library ER-Hoxb8 cells were stained intracellularly for S100A9 using a FITC-labelled antibody. Cas9-library ER-Hoxb8 day 3 monocytes with no or lower S100A9 expression were sorted as hits, the remaining cells served as reference cells. ( C ) Data was analysed using the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) software for identification of enriched guide RNAs in the hit sample. Corresponding genes were rank-ordered by robust rank aggregation (RRA) scores. The list states the top 20 genes according to RRA scores, arranged after the number of guides that are enriched in the hit sample. See also and . Figure 1—source data 1. Gene summary of Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MaGECK) analysis.

Article Snippet: Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) , was performed as described ( ).

Techniques: Genome Wide, Expressing, Purification, Next-Generation Sequencing, Staining, CRISPR, Knock-Out, Software

Journal: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet: Cell lines generated by CRISPR/Cas9 (A: PHF8, CSRP1, and HAND1 KO, dark grey bars = candidates from Genome-Scale CRISPR/Cas9 Knockout (GeCKO) screen, B: ATF3, STAT3, KLF5, IRF7, and C/EBPβ KO, light grey bars = candidates from previous studies) were compared to a non-targeting CRISPR/Cas9 control cell line. Cell lines generated from transgenic mice (C/EBPδ KO, black bars = hit from GeCKO screen) were compared to wildtype (WT) counterparts. All cells were differentiated to until day 2 or day 3 and relative S100a8 and S100a9 mRNA levels were analysed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Red lines indicate relative s100 level of controls (WT). Values are the means ± SEM of four experiments. *p<0.05, **p<0.01, ***p<0.001 by two-tailed Student’s t test.

Article Snippet: Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) , was performed as described ( ).

Techniques: Generated, CRISPR, Knock-Out, Control, Transgenic Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet:

Article Snippet: Amplification of mouse CRISPR Knockout pooled library (GeCKO v2) in lentiGuide-Puro plasmid, purchased from AddGene (#1000000053) , was performed as described ( ).

Techniques: Subcloning, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Recombinant, Control, Genome Wide, CRISPR, Expressing, Plasmid Preparation, Marker, Sequencing, Cloning, Library Amplification, Mutagenesis, In Vitro, In Vivo, Purification, Protease Inhibitor, Immunoprecipitation, Protein Extraction, Isolation, Software

Journal: Nature Communications

Article Title: ASPSCR1::TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma

doi: 10.1038/s41467-023-37049-z

Figure Lengend Snippet: a Schematic illustration of functional analysis in ASPS cells with or without ASPSCR1::TFE3 expression. b ASPSCR1::TFE3 expression in ASPS17 and ASPS25 was compared with their null counterparts at the transcriptional level (top) ( n = 3 per group) and protein levels (bottom, two representative immunoblots from eight independent experiments). c Loss of the ASPSCR1::TFE3 protein in ASPS null cells exhibited by immunofluorescence using the anti-FLAG antibody. Scale bar: 20 µm. Experiments represent five biological replicates. d Cell proliferation of mouse ASPS17 and ASPS null cells (left, top), and human ASPS-KY cells with siRNA treatment (left, bottom) ( n = 4 per group). ns: no significance. The efficiency of ASPSCR1::TFE3 knockdown is shown at transcriptional ( n = 4 per group) and protein levels (right, representative immunoblots from five independent experiments). e Suppression of tumor development by loss of ASPSCR1::TFE3 expression. Average tumor volumes with SD are shown in the recipient transplanted with ASPS17 and ASPS null cells ( n = 6 mice/12 independent tumors per group). The tumor volume was measured using 2 tumors per mouse. f Histology of transplanted area with ASPS17 and ASPS null cells 4 days after transplantation. Hematoxylin and eosin (HE) staining and immunohistochemistry with indicated antibodies. Scale bar: 50 µm. Experiments represent three biological replicates. g Gene set enrichment analysis (GSEA) showing enrichment of VEGF and exocytic vesicle pathways between ASPS17 and ASPS null cells. Normalized enrichment scores (NES), nominal p -values, and FDR q -values are indicated (left). The p -value is computed through the two-sided permutation test ( n = 1000 randomizations) adjusted the Benjamini-Hochberg procedure. Quantitative RT-PCR (qRT-PCR) showing downregulation of Rab27a, Sytl2, Pdgfb , and Vwf in ASPS null cells while Myh9 expression was increased ( n = 3 per group). h GSEA showing enrichment of VEGFA and pigment granule pathways by comparing human ASPS-KY cells with and without knockdown of ASPSCR1::TFE3 (left). Downregulation of RAB27A, SYTL2, PDGFB , and VWF is shown ( n = 3 per group). Statistical analyses in ( b , d , e , g , h ) were performed by two-sided Student’s t -test and *marks adjusted p -value < 0.05, **marks adjusted p -value < 0.01 and ***marks adjusted p -value < 0.001. The data presented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: 397 gRNAs for enhancers without ASPSCR1::TFE3 binding and 1000 non-target gRNAs ( https://www.addgene.org/pooled-library/zhang-mouse-gecko-v2/ ) were designed as negative controls.

Techniques: Functional Assay, Expressing, Western Blot, Immunofluorescence, Knockdown, Transplantation Assay, Staining, Immunohistochemistry, Quantitative RT-PCR

Journal: Nature Communications

Article Title: ASPSCR1::TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma

doi: 10.1038/s41467-023-37049-z

Figure Lengend Snippet: a Suppression of in vivo tumor growth by JQ1 treatment. Growth curves of the transplanted tumors treated with JQ1 or vehicle and the experimental schedule are shown (left) ( n = 5 mice/10 independent tumors per group). The tumor volume was measured using 2 tumors per mouse. Gross appearances of tumors resected on day 17 (right). b Immunohistological examination of the tumor samples treated with JQ1 or vehicle. Anti-Cd31 (endothel), anti-Pdgfrb (pericyte) and anti-FLAG (tumor cell) were used. Cd31- and Pdgfrb-positive areas were measured and quantitated using the image J software. Positive areas were normalized by the number of FLAG-positive cells (right) ( n = 17 and 5 independent areas for Cd31 and Pdgfrb, respectively). scale bar: 50 µm. Experiments represent three biological replicates. c GSEA showing PDGF-pathway enrichment by comparing vehicle-treated ASPS and ASPS treated with 0.5 µM JQ1 for 48 h gene expression signatures. The p -value is computed through the two-sided permutation test ( n = 1000 randomizations) adjusted the Benjamini-Hochberg procedure. d Ingenuity pathway analysis (IPA) exhibiting multiple genetic pathways including angiogenesis/vasculogenesis (described in red) by JQ1 treatment. p -value was calculated using Fischer’s exact test. e Composite plots showing a significant reduction in Brd4-binding signals around ASPSCR1::TFE3-binding peaks by JQ1 treatment (left). Heat maps showing ASPSCR1::TFE3 and Brd4 with or without JQ1 treatment, in murine ASPS17 cells (right). f Venn diagram showing overlapping and distinct Brd4-binding peaks in ASPS17 cells treated with JQ1 and vehicle (top). GREAT using 23,924 genetic loci specific for vehicle-treated ASPS17 showing the enrichment of angiogenesis-related pathways (bottom). p -values are calculated using a binominal test. g ChIP-seq track at Rab27a, Sytl2, Pdgfb, Vwf, Myh9 , and Neat1 genomic loci with ASPSCR1::TFE3-binding peaks in ASPS17 cells. Significant reduction of Brd4 signals at Rab27a, Sytl2, Pdgfb , and Vwf loci. ASPSCR1::TFE3 signals remained unchanged by JQ1 treatment. h qRT-PCR showing downregulation of Rab27a, Sytl2, Pdgfb , and Vwf in ASPS17 treated with JQ1, with the expression of Myh9 and Neat1 remaining unchanged ( n = 3 per group). Statistical analyses in ( a , b , h ) were performed by two-sided Student’s t -test and *marks adjusted p -value < 0.05, **marks adjusted p -value < 0.01 and ***marks adjusted p- value < 0.001. ns: no significance. The data presented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: 397 gRNAs for enhancers without ASPSCR1::TFE3 binding and 1000 non-target gRNAs ( https://www.addgene.org/pooled-library/zhang-mouse-gecko-v2/ ) were designed as negative controls.

Techniques: In Vivo, Software, Gene Expression, Binding Assay, ChIP-sequencing, Quantitative RT-PCR, Expressing

Journal: Nature immunology

Article Title: Chemosensor receptors are lipid-detecting regulators of macrophage function in cancer.

doi: 10.1038/s41590-025-02191-x

Figure Lengend Snippet: Fig. 2 | CRISPR–Cas9 screening of primary mouse macrophages to identify TAM regulators. a, Experimental scheme of genome-wide CRISPR–Cas9 Knockout GecKO v2 Library B screening in primary murine macrophages. b, Representative plot of backbone (LGP) or library-B-infected macrophages exposed or not to conditioned media from Pten−/−Trp53−/− tumor cells. c, Two independent experiments were performed. Graphs show the correlations between the distribution of the guides found in the CD206−MHCII+ population (MHCII) and in the CD206brightMHCII− population (CD206) from the two experiments. Lib1, library 1; Lib2, library 2. d, Volcano plot showing genes related to the differentially enriched sgRNA guides from CD206−MHCII+ versus CD206brightMHCII− cells. Negative regulators of the CD206brightMHCII− population are shown in light blue, and positive regulators are shown in red. log2FC ± 0.56, P < 0.005. Statistical analyses and comparisons from NGS output

Article Snippet: The mouse GeCKO v2 Library B (Addgene) was used: this library consists of 62,804 sgRNAs constructs, with three sgRNAs targeting each of the 20,661 genes of the mouse genome.

Techniques: CRISPR, Genome Wide, Knock-Out, Infection

Journal: Science Advances

Article Title: Cullin-5 deficiency orchestrates the tumor microenvironment to promote mammary tumor development through CREB1-CCL2 signaling

doi: 10.1126/sciadv.abq1395

Figure Lengend Snippet: ( A ) Tumor-free curves of Brca1 Flox11/Flox11 ;Wap-Cre or Brca1 Flox11/Flox11 ;MMTV-Cre mice with or without SB transposon insertion in SB mice. P ≤ 0.0001. ( B ) The SB transposon insertion sites and directions in the DNA sequence of Cul5 . ( C ) Cell growth of MEFs after infection with a lentivirus-packaged mouse GeCKOv2 sgRNA library. Scale bars, 50 μm. ( D ) Representative photographs of primary tumors formed in nude mice transplanted with MEFs (passages 1 and 16) in fat pads. ( E ) Statistical analysis of sgRNA numbers that represent candidate genes appearing in both in vivo and in vitro screenings. ( F ) Cul5 and Fbxo9 were common candidate genes in CRISPR-Cas9 and SB screening. ( G ) The Cancer Genome Atlas statistics showing the mRNA expression level of CUL5 in normal mammary tissues, primary tumors, and metastatic tumors from patients with breast cancer. P ≤ 0.001. ( H ) Kaplan-Meier plot showing the relationships between CUL5 expression and the probability of survival in 1010 human breast cancer patients (506 lower CUL5 and 504 higher CUL5).

Article Snippet: First, we packaged the lentivirus with a loss-of-function mouse GeCKOv2 sgRNA library (Addgene, pooled library #1000000052), which included 123,666 sgRNAs targeting 20,611 genes, 4700 sgRNAs targeting 1175 microRNAs (miRNAs), and 1000 nontargeting sgRNAs as controls ( ).

Techniques: Sequencing, Infection, In Vivo, In Vitro, CRISPR, Expressing

Journal: Science advances

Article Title: Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.

doi: 10.1126/sciadv.aaz6699

Figure Lengend Snippet: Fig. 5. PD-MK conditioned mESCs retain pluripotency. (A) Growth curve of mESC conditioned to Std, 2i, and PD-MK conditions. Error bars indicate SD (n = 3). (B) Per- centage of apoptotic cells of mESC conditioned to Std, 2i, and PD-MK conditions. Error bars indicate SD (n = 3). (C) Cell cycle distribution of mESCs conditioned to Std, 2i, and PD-MK conditions. (D) Immunofluorescence of pluripotency markers (NANOG, OCT4, and SSEA1) of mESCs conditioned to Std, 2i, and PD-MK conditions. The images show maximum intensity projections of stacks. Scale bar, 50 m. (E) Chimeric mice with black coat color generated from C57BL/6NCr ES cells conditioned to the PD-MK condition and then to the Std condition before injection into albino host embryos. Photo credit: T. Okamura (National Center for Global Health and Medicine).

Article Snippet: Infection of CRISPR lentivirus library Nanog, Trim28, and Dnmt3L KI cells were transduced with the Mouse CRISPR K/O Pooled Library (GeCKO v2; Addgene, #1000000052) (29) via spinfection as previously described.

Techniques: Immunofluorescence, Generated, Injection