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ascas12a crrna expression vector  (Addgene inc)


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    Addgene inc ascas12a crrna expression vector
    Ascas12a Crrna Expression Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ascas12a crrna expression vector/product/Addgene inc
    Average 93 stars, based on 9 article reviews
    ascas12a crrna expression vector - by Bioz Stars, 2026-03
    93/100 stars

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    A. Experimental workflow for double-gene deletion <t>AsCas12a</t> CRISPR screening. B. Scatter plot of median Log2 fold-change of crRNAs associated with the indicated target gene after Cas12a-mediated co-deletion of Rosa26 control (WT) or Ripk1 (Ripk1null). Fold-change is calculated between in vitro and in vivo timepoints. Targets preferentially depleted in WT (red), Ripk1null (beige), or both (blue), or targets preferentially enriched in Ripk1null (orange) are highlighted and have a P-value < 0.05 (see Methods). Also shown are Log2 fold-change for individual crRNAs (red bars) for significant hits overlaid on the distribution for all crRNAs. C. Select targets identified in (B) projected onto a schematic of the TNF signaling pathway in Ripk1 WT (top) and Ripk1null (bottom) cancer cells. Highlighted gene targets (non-opaque) are depleted in WT or enriched in Ripk1null tumors and illustrate inferred signaling bias for each genotype. D-E. Expression and quantitation of NF-kB and MAPK pathway proteins (n=2–3) (D) and NF-kB transcriptional reporter activity (representative of 3 independent experiments) (E) in WT or Ripk1null B16 cancer cells after treatment with 100 ng/ml murine TNF. F. CASP3 cleavage after TNF stimulation of TSA WT or Ripk1null cells for the indicated times under serum-free conditions. G. In vitro dose response of TNF-mediated killing with 1 ug/ml cycloheximide for 24 hours for WT or Ripk1null B16 and TSA cells measured by normalized viability (representative of 2–3 independent experiments). P-values for time course was determined by repeated measures ANOVA. For dose response and reporter assay, a non-linear model was fitted and significance determined by comparison to a reduced model using ANOVA.
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    A. Experimental workflow for double-gene deletion <t>AsCas12a</t> CRISPR screening. B. Scatter plot of median Log2 fold-change of crRNAs associated with the indicated target gene after Cas12a-mediated co-deletion of Rosa26 control (WT) or Ripk1 (Ripk1null). Fold-change is calculated between in vitro and in vivo timepoints. Targets preferentially depleted in WT (red), Ripk1null (beige), or both (blue), or targets preferentially enriched in Ripk1null (orange) are highlighted and have a P-value < 0.05 (see Methods). Also shown are Log2 fold-change for individual crRNAs (red bars) for significant hits overlaid on the distribution for all crRNAs. C. Select targets identified in (B) projected onto a schematic of the TNF signaling pathway in Ripk1 WT (top) and Ripk1null (bottom) cancer cells. Highlighted gene targets (non-opaque) are depleted in WT or enriched in Ripk1null tumors and illustrate inferred signaling bias for each genotype. D-E. Expression and quantitation of NF-kB and MAPK pathway proteins (n=2–3) (D) and NF-kB transcriptional reporter activity (representative of 3 independent experiments) (E) in WT or Ripk1null B16 cancer cells after treatment with 100 ng/ml murine TNF. F. CASP3 cleavage after TNF stimulation of TSA WT or Ripk1null cells for the indicated times under serum-free conditions. G. In vitro dose response of TNF-mediated killing with 1 ug/ml cycloheximide for 24 hours for WT or Ripk1null B16 and TSA cells measured by normalized viability (representative of 2–3 independent experiments). P-values for time course was determined by repeated measures ANOVA. For dose response and reporter assay, a non-linear model was fitted and significance determined by comparison to a reduced model using ANOVA.
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    A. Experimental workflow for double-gene deletion AsCas12a CRISPR screening. B. Scatter plot of median Log2 fold-change of crRNAs associated with the indicated target gene after Cas12a-mediated co-deletion of Rosa26 control (WT) or Ripk1 (Ripk1null). Fold-change is calculated between in vitro and in vivo timepoints. Targets preferentially depleted in WT (red), Ripk1null (beige), or both (blue), or targets preferentially enriched in Ripk1null (orange) are highlighted and have a P-value < 0.05 (see Methods). Also shown are Log2 fold-change for individual crRNAs (red bars) for significant hits overlaid on the distribution for all crRNAs. C. Select targets identified in (B) projected onto a schematic of the TNF signaling pathway in Ripk1 WT (top) and Ripk1null (bottom) cancer cells. Highlighted gene targets (non-opaque) are depleted in WT or enriched in Ripk1null tumors and illustrate inferred signaling bias for each genotype. D-E. Expression and quantitation of NF-kB and MAPK pathway proteins (n=2–3) (D) and NF-kB transcriptional reporter activity (representative of 3 independent experiments) (E) in WT or Ripk1null B16 cancer cells after treatment with 100 ng/ml murine TNF. F. CASP3 cleavage after TNF stimulation of TSA WT or Ripk1null cells for the indicated times under serum-free conditions. G. In vitro dose response of TNF-mediated killing with 1 ug/ml cycloheximide for 24 hours for WT or Ripk1null B16 and TSA cells measured by normalized viability (representative of 2–3 independent experiments). P-values for time course was determined by repeated measures ANOVA. For dose response and reporter assay, a non-linear model was fitted and significance determined by comparison to a reduced model using ANOVA.

    Journal: Immunity

    Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

    doi: 10.1016/j.immuni.2022.03.007

    Figure Lengend Snippet: A. Experimental workflow for double-gene deletion AsCas12a CRISPR screening. B. Scatter plot of median Log2 fold-change of crRNAs associated with the indicated target gene after Cas12a-mediated co-deletion of Rosa26 control (WT) or Ripk1 (Ripk1null). Fold-change is calculated between in vitro and in vivo timepoints. Targets preferentially depleted in WT (red), Ripk1null (beige), or both (blue), or targets preferentially enriched in Ripk1null (orange) are highlighted and have a P-value < 0.05 (see Methods). Also shown are Log2 fold-change for individual crRNAs (red bars) for significant hits overlaid on the distribution for all crRNAs. C. Select targets identified in (B) projected onto a schematic of the TNF signaling pathway in Ripk1 WT (top) and Ripk1null (bottom) cancer cells. Highlighted gene targets (non-opaque) are depleted in WT or enriched in Ripk1null tumors and illustrate inferred signaling bias for each genotype. D-E. Expression and quantitation of NF-kB and MAPK pathway proteins (n=2–3) (D) and NF-kB transcriptional reporter activity (representative of 3 independent experiments) (E) in WT or Ripk1null B16 cancer cells after treatment with 100 ng/ml murine TNF. F. CASP3 cleavage after TNF stimulation of TSA WT or Ripk1null cells for the indicated times under serum-free conditions. G. In vitro dose response of TNF-mediated killing with 1 ug/ml cycloheximide for 24 hours for WT or Ripk1null B16 and TSA cells measured by normalized viability (representative of 2–3 independent experiments). P-values for time course was determined by repeated measures ANOVA. For dose response and reporter assay, a non-linear model was fitted and significance determined by comparison to a reduced model using ANOVA.

    Article Snippet: Fluorescently labeled anti-mouse antibodies against CD45 (104), I-A/I-E (M5/114.15.2), F4/80 (BM8), CD11b (M1/70), CD11c (N418), CD103 (2E7), PD-L1 (B7-H1), Ly6C (HK1.4), Ly6G (1A8), PD-1 (Rmp1–30), CD3 (145–2C11), B220 (RA3–6B2), and NKP46 (29A1.4) were obtained from BioLegend; CD8 (53–6.7), CD4 (rm4–5), NK1.1 (PK136), and F4/80 (BM8) from eBioscience; CD11c (HL3) and TCRb (H57–597) from BD Biosciences; CD11b (M1/70), Arg1 (A1exF5), and Granzyme B (GB11) from Thermo Fisher Scientific. . Double-gene deletion CRISPR library assembly For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

    Techniques: CRISPR, Control, In Vitro, In Vivo, Expressing, Quantitation Assay, Activity Assay, Reporter Assay, Comparison

    KEY RESOURCES TABLE

    Journal: Immunity

    Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

    doi: 10.1016/j.immuni.2022.03.007

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Fluorescently labeled anti-mouse antibodies against CD45 (104), I-A/I-E (M5/114.15.2), F4/80 (BM8), CD11b (M1/70), CD11c (N418), CD103 (2E7), PD-L1 (B7-H1), Ly6C (HK1.4), Ly6G (1A8), PD-1 (Rmp1–30), CD3 (145–2C11), B220 (RA3–6B2), and NKP46 (29A1.4) were obtained from BioLegend; CD8 (53–6.7), CD4 (rm4–5), NK1.1 (PK136), and F4/80 (BM8) from eBioscience; CD11c (HL3) and TCRb (H57–597) from BD Biosciences; CD11b (M1/70), Arg1 (A1exF5), and Granzyme B (GB11) from Thermo Fisher Scientific. . Double-gene deletion CRISPR library assembly For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

    Techniques: Control, Virus, Recombinant, Protease Inhibitor, Membrane, Lysis, Transfection, Luciferase, Cell Viability Assay, Mutagenesis, Staining, Sequencing, RNA Sequencing Assay, Derivative Assay, Expressing, CRISPR, Plasmid Preparation, Retroviral, Scaffolding, Software

    KEY RESOURCES TABLE

    Journal: Immunity

    Article Title: The Interferon-Stimulated Gene RIPK1 Regulates Cancer Cell Intrinsic and Extrinsic Resistance to Immune Checkpoint Blockade

    doi: 10.1016/j.immuni.2022.03.007

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Fluorescently labeled anti-mouse antibodies against CD45 (104), I-A/I-E (M5/114.15.2), F4/80 (BM8), CD11b (M1/70), CD11c (N418), CD103 (2E7), PD-L1 (B7-H1), Ly6C (HK1.4), Ly6G (1A8), PD-1 (Rmp1–30), CD3 (145–2C11), B220 (RA3–6B2), and NKP46 (29A1.4) were obtained from BioLegend; CD8 (53–6.7), CD4 (rm4–5), NK1.1 (PK136), and F4/80 (BM8) from eBioscience; CD11c (HL3) and TCRb (H57–597) from BD Biosciences; CD11b (M1/70), Arg1 (A1exF5), and Granzyme B (GB11) from Thermo Fisher Scientific. . Double-gene deletion CRISPR library assembly For double-gene deletion CRISPR screening, a dual-crRNA library was cloned into a AsCas12a crRNA expression vector pRG212 (EFS-GFP-P2A-Neo-U6-crRNA, Addgene #149722).

    Techniques: Control, Virus, Recombinant, Protease Inhibitor, Membrane, Lysis, Transfection, Luciferase, Cell Viability Assay, Mutagenesis, Staining, Sequencing, RNA Sequencing Assay, Derivative Assay, Expressing, CRISPR, Plasmid Preparation, Retroviral, Scaffolding, Software

    Journal: iScience

    Article Title: Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia

    doi: 10.1016/j.isci.2022.105139

    Figure Lengend Snippet:

    Article Snippet: The total 4 crRNAs were cloned in a crRNA array of the pRG212 lentiviral expression vector (Addgene: 149,722).

    Techniques: Recombinant, Reverse Transcription, Magnetic Beads, Protease Inhibitor, Cell Viability Assay, Cloning, Purification, Isolation, Sequencing, Software