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pcc 01  (Addgene inc)


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    Addgene inc pcc 01
    Pcc 01, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pcc+01/pCC_01+-+hU6-BsmBI-sgRNA(E%2BF)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE+(Plasmid+%23139086)/pmc11825950-416-20-21
    Average 93 stars, based on 13 article reviews
    pcc 01 - by Bioz Stars, 2026-09
    93/100 stars

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    Related Articles

    other:

    Article Title: Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.
    Article Snippet: We then synthesized a human codon-optimized mStayGold124 as a gBlock (IDT) and cloned it into pLentiSpCas9guide using AgeI and BamHI restriction sites and T4 ligase (New England Biolabs); we termed this plasmid lentiGuideFEmSG-Puro (Addgene, 226522).

    Modification:

    Article Title: Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system
    Article Snippet: .. pCC_01 and pCC_09 (Addgene #139094) were modified to incorporate Capture Sequences 1 and 2 at the 3′ end of guide sequences. ..

    CRISPR:

    Article Title: Paired CRISPR screens to map gene regulation in cis and trans
    Article Snippet: .. The TFome-wide CRISPR library was cloned into the pCC_01 all-in-one lentiviral vector (Addgene 139086) with an optimized Cas9 scaffold . .. Cloned libraries were amplified by electroporation into Endura electrocompetent cells (Lucigen).

    Clone Assay:

    Article Title: Paired CRISPR screens to map gene regulation in cis and trans
    Article Snippet: .. The TFome-wide CRISPR library was cloned into the pCC_01 all-in-one lentiviral vector (Addgene 139086) with an optimized Cas9 scaffold . .. Cloned libraries were amplified by electroporation into Endura electrocompetent cells (Lucigen).

    Article Title: Paired CRISPR screens to map gene regulation in cis and trans
    Article Snippet: The saturation mutagenesis CRISPR library were cloned into the backbone lentiCRISPRv2 (Addgene 52961) containing Cas9, a guide RNA cassette and a puro resistant cassette . .. Individual gRNAs were cloned into the pCC_01 all-in-one lentiviral vector (Addgene 139086) with an optimized Cas9 scaffold . ..

    Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers
    Article Snippet: .. To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro). .. We then synthesized a human codon-optimized mStayGold as a gBlock (IDT) and cloned it into pLentiSpCas9guide using AgeI and BamHI restriction sites and T4 ligase (New England Biolabs); we termed this plasmid lentiGuideFE-mSG-Puro (Addgene, 226522).

    Article Title: Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.
    Article Snippet: After 5 days, we sorted transduced cells (mKate2-positive cells) using a Sony SH800 cell sorter. .. To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned theU6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_00393 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro). .. We then synthesized a human codon-optimized mStayGold124 as a gBlock (IDT) and cloned it into pLentiSpCas9guide using AgeI and BamHI restriction sites and T4 ligase (New England Biolabs); we termed this plasmid lentiGuideFEmSG-Puro (Addgene, 226522).

    Article Title: Paired CRISPR screens to map gene regulation in cis and trans
    Article Snippet: .. For individual gRNA, standard desalted short oligonucleotides (IDT) were annealed and cloned into the pCC_01 (Addgene 139086) . ..

    Plasmid Preparation:

    Article Title: Paired CRISPR screens to map gene regulation in cis and trans
    Article Snippet: .. The TFome-wide CRISPR library was cloned into the pCC_01 all-in-one lentiviral vector (Addgene 139086) with an optimized Cas9 scaffold . .. Cloned libraries were amplified by electroporation into Endura electrocompetent cells (Lucigen).

    Article Title: Paired CRISPR screens to map gene regulation in cis and trans
    Article Snippet: The saturation mutagenesis CRISPR library were cloned into the backbone lentiCRISPRv2 (Addgene 52961) containing Cas9, a guide RNA cassette and a puro resistant cassette . .. Individual gRNAs were cloned into the pCC_01 all-in-one lentiviral vector (Addgene 139086) with an optimized Cas9 scaffold . ..

    Article Title: Comprehensive dissection of cis- regulatory elements in a 2.8 Mb topologically associated domain in six human cancers
    Article Snippet: .. To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned the U6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_003 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro). .. We then synthesized a human codon-optimized mStayGold as a gBlock (IDT) and cloned it into pLentiSpCas9guide using AgeI and BamHI restriction sites and T4 ligase (New England Biolabs); we termed this plasmid lentiGuideFE-mSG-Puro (Addgene, 226522).

    Article Title: Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.
    Article Snippet: After 5 days, we sorted transduced cells (mKate2-positive cells) using a Sony SH800 cell sorter. .. To generate CRISPRi guide plasmids with mStayGold (a green fluorescent protein), we first cloned theU6-sgRNA(F + E) cassette from pCC_01 (Addgene 139086) into pLentiRNAGuide_00393 (Addgene 192505) using PacI and NheI restriction sites and termed the plasmid pLentiSpCas9guide (GFP-P2A-Puro). .. We then synthesized a human codon-optimized mStayGold124 as a gBlock (IDT) and cloned it into pLentiSpCas9guide using AgeI and BamHI restriction sites and T4 ligase (New England Biolabs); we termed this plasmid lentiGuideFEmSG-Puro (Addgene, 226522).



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    A. Immunoblot of KRT20 and GAPDH proteins in LS180 treated with MRK60 (2 µM) and indicated concentrations of JQAD1 (0.4 - 10 µM). B. Histone-enriched immunoblot of H3K27ac and H3 in HT-29 treated with MRK60 (5 µM) +/- JQAD1 (10 µM). C. Quantification of GFP+ percentage in HT29 KRT20-GFP reporter cell line treated with MRK60 (5 µM) +/- JQAD1 (10 µM). D. Ranked log 2 fold change of sgRNA distribution in GFP high (perturbation promotes differentiation) and mKate2 high (perturbation promotes stem cell) sorted cell fractions of published <t>CRISPR-Cas9</t> screen targeting epigenetic regulators (78 genes 542 sgRNAs) using HT29 SOX9-mKate2/KRT20-GFP reporter cell line 16 ; select perturbations indicated in colored circles. E. Schematic of scRNA-seq experiment in patient-derived CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1 combination (left). UMAPs of scRNA-seq colored by treatment group (middle) or cell type (right). F. Proportion of cell types in CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1. G. Trajectory analysis of colonocytes in scRNA-seq experiment colored by treatment group. H. Expression of colonocyte differentiation signatures along drug response trajectory. Density plot shows histogram of cells in different treatment along pseudotime. ** P = 0.0042; *** P = 0.0002 I. Violin plot of colonocyte differentiation gene signature in scRNA-seq experiment by treatment groups. **** P = 7.0 x 10 -6 (left), **** P = 8.4 x 10 -8 (right) J. Violin plot of gene signatures associated with MRK60-induced gains in H3K27ac bound by HDAC1/2 in scRNA-seq experiment by treatment groups. **** P = 5.8 x 10 -10 (left), **** P = 2.1 x 10 -5 (right) K. Integrative heatmap of expression changes in stem cell and differentiation genes associated with H3K27ac, H3K9ac, and H4K8ac in bulk and single-cell RNA data sets with MRK60 and/or JQAD1.
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    A. Immunoblot of KRT20 and GAPDH proteins in LS180 treated with MRK60 (2 µM) and indicated concentrations of JQAD1 (0.4 - 10 µM). B. Histone-enriched immunoblot of H3K27ac and H3 in HT-29 treated with MRK60 (5 µM) +/- JQAD1 (10 µM). C. Quantification of GFP+ percentage in HT29 KRT20-GFP reporter cell line treated with MRK60 (5 µM) +/- JQAD1 (10 µM). D. Ranked log 2 fold change of sgRNA distribution in GFP high (perturbation promotes differentiation) and mKate2 high (perturbation promotes stem cell) sorted cell fractions of published <t>CRISPR-Cas9</t> screen targeting epigenetic regulators (78 genes 542 sgRNAs) using HT29 SOX9-mKate2/KRT20-GFP reporter cell line 16 ; select perturbations indicated in colored circles. E. Schematic of scRNA-seq experiment in patient-derived CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1 combination (left). UMAPs of scRNA-seq colored by treatment group (middle) or cell type (right). F. Proportion of cell types in CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1. G. Trajectory analysis of colonocytes in scRNA-seq experiment colored by treatment group. H. Expression of colonocyte differentiation signatures along drug response trajectory. Density plot shows histogram of cells in different treatment along pseudotime. ** P = 0.0042; *** P = 0.0002 I. Violin plot of colonocyte differentiation gene signature in scRNA-seq experiment by treatment groups. **** P = 7.0 x 10 -6 (left), **** P = 8.4 x 10 -8 (right) J. Violin plot of gene signatures associated with MRK60-induced gains in H3K27ac bound by HDAC1/2 in scRNA-seq experiment by treatment groups. **** P = 5.8 x 10 -10 (left), **** P = 2.1 x 10 -5 (right) K. Integrative heatmap of expression changes in stem cell and differentiation genes associated with H3K27ac, H3K9ac, and H4K8ac in bulk and single-cell RNA data sets with MRK60 and/or JQAD1.
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    Image Search Results


    A. Immunoblot of KRT20 and GAPDH proteins in LS180 treated with MRK60 (2 µM) and indicated concentrations of JQAD1 (0.4 - 10 µM). B. Histone-enriched immunoblot of H3K27ac and H3 in HT-29 treated with MRK60 (5 µM) +/- JQAD1 (10 µM). C. Quantification of GFP+ percentage in HT29 KRT20-GFP reporter cell line treated with MRK60 (5 µM) +/- JQAD1 (10 µM). D. Ranked log 2 fold change of sgRNA distribution in GFP high (perturbation promotes differentiation) and mKate2 high (perturbation promotes stem cell) sorted cell fractions of published CRISPR-Cas9 screen targeting epigenetic regulators (78 genes 542 sgRNAs) using HT29 SOX9-mKate2/KRT20-GFP reporter cell line 16 ; select perturbations indicated in colored circles. E. Schematic of scRNA-seq experiment in patient-derived CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1 combination (left). UMAPs of scRNA-seq colored by treatment group (middle) or cell type (right). F. Proportion of cell types in CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1. G. Trajectory analysis of colonocytes in scRNA-seq experiment colored by treatment group. H. Expression of colonocyte differentiation signatures along drug response trajectory. Density plot shows histogram of cells in different treatment along pseudotime. ** P = 0.0042; *** P = 0.0002 I. Violin plot of colonocyte differentiation gene signature in scRNA-seq experiment by treatment groups. **** P = 7.0 x 10 -6 (left), **** P = 8.4 x 10 -8 (right) J. Violin plot of gene signatures associated with MRK60-induced gains in H3K27ac bound by HDAC1/2 in scRNA-seq experiment by treatment groups. **** P = 5.8 x 10 -10 (left), **** P = 2.1 x 10 -5 (right) K. Integrative heatmap of expression changes in stem cell and differentiation genes associated with H3K27ac, H3K9ac, and H4K8ac in bulk and single-cell RNA data sets with MRK60 and/or JQAD1.

    Journal: bioRxiv

    Article Title: Chemical perturbations impacting histone acetylation govern colorectal cancer differentiation

    doi: 10.1101/2024.12.06.626451

    Figure Lengend Snippet: A. Immunoblot of KRT20 and GAPDH proteins in LS180 treated with MRK60 (2 µM) and indicated concentrations of JQAD1 (0.4 - 10 µM). B. Histone-enriched immunoblot of H3K27ac and H3 in HT-29 treated with MRK60 (5 µM) +/- JQAD1 (10 µM). C. Quantification of GFP+ percentage in HT29 KRT20-GFP reporter cell line treated with MRK60 (5 µM) +/- JQAD1 (10 µM). D. Ranked log 2 fold change of sgRNA distribution in GFP high (perturbation promotes differentiation) and mKate2 high (perturbation promotes stem cell) sorted cell fractions of published CRISPR-Cas9 screen targeting epigenetic regulators (78 genes 542 sgRNAs) using HT29 SOX9-mKate2/KRT20-GFP reporter cell line 16 ; select perturbations indicated in colored circles. E. Schematic of scRNA-seq experiment in patient-derived CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1 combination (left). UMAPs of scRNA-seq colored by treatment group (middle) or cell type (right). F. Proportion of cell types in CRC organoids treated with DMSO, MRK60, or MRK60+JQAD1. G. Trajectory analysis of colonocytes in scRNA-seq experiment colored by treatment group. H. Expression of colonocyte differentiation signatures along drug response trajectory. Density plot shows histogram of cells in different treatment along pseudotime. ** P = 0.0042; *** P = 0.0002 I. Violin plot of colonocyte differentiation gene signature in scRNA-seq experiment by treatment groups. **** P = 7.0 x 10 -6 (left), **** P = 8.4 x 10 -8 (right) J. Violin plot of gene signatures associated with MRK60-induced gains in H3K27ac bound by HDAC1/2 in scRNA-seq experiment by treatment groups. **** P = 5.8 x 10 -10 (left), **** P = 2.1 x 10 -5 (right) K. Integrative heatmap of expression changes in stem cell and differentiation genes associated with H3K27ac, H3K9ac, and H4K8ac in bulk and single-cell RNA data sets with MRK60 and/or JQAD1.

    Article Snippet: The pCC_01 - hU6-BsmBI-sgRNA(E + F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE plasmid (Addgene #139086; RRID:Addgene_139086) was used for CRISPR knockout.

    Techniques: Western Blot, CRISPR, Derivative Assay, Expressing

    A. Venn diagram showing genes bound by HDAC1/2 and MRK60, upregulated by MRK60, and gained H3K27ac upon MRK60 treatment. B. Schematic diagram showing CRISPR-Cas9 genetic screen in HT29 KRT20-GFP reporter cells. C. Ranked normalized Z-scored log 2 fold change (LFC) plots of sgRNA targeting 27 genes in HT29 KRT20-GFP reporter screen with respect to viability (top) and GFP/KRT20 (bottom) outputs D. mRNA and (E) protein expression of KRT20 and DAPK3 in HT115 cells engineered to suppress DAPK3 by CRISPRi knockdown treated with 2µM MRK60 for 72 hours. F. Integrative Genomic Viewer (IGV) snapshot depicting HDAC1/2 binding, H3K27ac, H3K9ac, and H4K8ac CUT&RUN peaks and ATAC-seq profiles at DAPK3 genomic locus. G. Immunoblots of Cdx2 CreERT2 ; Apc f/f ; R26 tdT adenoma mouse organoids treated with 5µM MRK60 and indicated doses of Ruxolitinib at 72 hours. H. Schematic diagram showing a proposed differentiation mechanism mediated by HDAC1/2 and H3K27ac

    Journal: bioRxiv

    Article Title: Chemical perturbations impacting histone acetylation govern colorectal cancer differentiation

    doi: 10.1101/2024.12.06.626451

    Figure Lengend Snippet: A. Venn diagram showing genes bound by HDAC1/2 and MRK60, upregulated by MRK60, and gained H3K27ac upon MRK60 treatment. B. Schematic diagram showing CRISPR-Cas9 genetic screen in HT29 KRT20-GFP reporter cells. C. Ranked normalized Z-scored log 2 fold change (LFC) plots of sgRNA targeting 27 genes in HT29 KRT20-GFP reporter screen with respect to viability (top) and GFP/KRT20 (bottom) outputs D. mRNA and (E) protein expression of KRT20 and DAPK3 in HT115 cells engineered to suppress DAPK3 by CRISPRi knockdown treated with 2µM MRK60 for 72 hours. F. Integrative Genomic Viewer (IGV) snapshot depicting HDAC1/2 binding, H3K27ac, H3K9ac, and H4K8ac CUT&RUN peaks and ATAC-seq profiles at DAPK3 genomic locus. G. Immunoblots of Cdx2 CreERT2 ; Apc f/f ; R26 tdT adenoma mouse organoids treated with 5µM MRK60 and indicated doses of Ruxolitinib at 72 hours. H. Schematic diagram showing a proposed differentiation mechanism mediated by HDAC1/2 and H3K27ac

    Article Snippet: The pCC_01 - hU6-BsmBI-sgRNA(E + F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE plasmid (Addgene #139086; RRID:Addgene_139086) was used for CRISPR knockout.

    Techniques: CRISPR, Expressing, Knockdown, Binding Assay, Western Blot