plix403 ccdb blast  (Addgene inc)

Journal: Nature Communications

Article Title: Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

doi: 10.1038/s41467-024-46285-w

Figure Lengend Snippet: a Schematic showing integration of T2A-GFP-P2A-Neo reporter cassette downstream of SOX9 ’s 3rd exon. Stop codon indicated by red box. b Light microscopy (top) and histogram (bottom) showing distribution of GFP fluorescence intensity of the LS180 SOX9-GFP and its parental cells. c Percentage of GFP+ cells in parental and LS180 SOX9-GFP upon NTC or SOX9 knockdown. d Schematic of 76 sgRNA CRISPR-Cas9 and 161 shRNAs screens targeting 8 genes in the LS180 SOX9-GFP line. e Ranked log 2 FC plot of shRNA and CRISPR-Cas9 screens in LS180 SOX9 line comparing the top and bottom 2.5% GFP positive fractions. SOX9 and EGFP shRNAs or sgRNAs are in blue and green, respectively. f Ranked log 2 FC plot of shRNA and CRISPR-Cas9 screens in LS180 SOX9 line comparing the top and bottom 2.5% GFP positive fractions on days 3 and 7 following library infection. g Volcano plots of CRISPR-Cas9 screen targeting epigenetic regulators (542 sgRNAs targeting 78 genes) in HT29 SOX9-mKate2 cell-line comparing the indicated 4 mKate2 fractions on day 7. The x-axis shows the Z score of gene-level median log 2 FC of all sgRNAs while the y-axis shows the gene-level p -values generated by MaGeCK MLE. h Beta score of each gene in CRISPR-Cas9 screen targeting epigenetic regulators in HT29 SOX9 -mKate2 line (3 technical replicates) comparing the top and bottom 25% mKate2+ fractions. i Rank sum of each gene in CRISPR-Cas9 screen targeting epigenetic regulators in HT29 SOX9 -mKate2 line comparing top and bottom 25% of mKate2+ fractions. Boxplots show distribution of sgRNAs per gene. Probability density plots showing the distribution of enriched (green), depleted (red), and all other sgRNA (gray) rank sums (right panel).

Article Snippet: (Addgene #158560) was used for doxycycline-inducible SOX9 overexpression.

Techniques: Light Microscopy, Fluorescence, Knockdown, CRISPR, shRNA, Infection, Generated

Journal: Nature Communications

Article Title: Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

doi: 10.1038/s41467-024-46285-w

Figure Lengend Snippet: a H&E as well as Sox9, Krt20, and tdT immunohistochemistry in normal and Apc KO mouse colon; scale bar = 100 µM. b Schematic showing integration of T2A-GFP-P2A-Neo reporter cassette downstream of KRT20 ’s 8th exon. Stop codon indicated by red box. c Ranked log 2 FC plot of 161 shRNAs and 76 sgRNAs CRISPR screens in HT29 KRT20 -GFP cells comparing the top and bottom 2.5% GFP+ fractions. SOX9, KRT20, and EGFP shRNAs or sgRNAs are indicated by blue, yellow, and green, respectively. d Ranked log 2 FC plot of CRISPR-Cas9 screen in HT29 KRT20 -GFP cells comparing the top and bottom 2.5% GFP+ fractions on days 4 and 7 following library infection. e Volcano plots of CRISPR-Cas9 screen targeting epigenetic regulators (542 sgRNAs targeting 78 genes) in HT29 KRT20 -GFP cell-line comparing the indicated 4 GFP fractions on day 7. The x-axis shows the Z score of gene-level median log 2 FC of all sgRNAs while the y-axis shows the gene-level, adjusted p -values generated by MaGeCK MLE. f Beta score of each gene in CRISPR-Cas9 screen targeting epigenetic regulators in HT29 KRT20 -GFP line (3 technical replicates) comparing the top and bottom 25% GFP+ fractions. g Rank sum of each gene in CRISPR-Cas9 screen targeting epigenetic regulators in HT29 KRT20 -GFP line comparing top and bottom 25% of GFP+ fractions. Boxplots show distribution of sgRNAs per gene. Probability density plots show distribution of enriched (green), depleted (red), and all other sgRNA (gray) rank sums (right panel).

Article Snippet: (Addgene #158560) was used for doxycycline-inducible SOX9 overexpression.

Techniques: Immunohistochemistry, CRISPR, Infection, Generated

Journal: Nature Communications

Article Title: Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

doi: 10.1038/s41467-024-46285-w

Figure Lengend Snippet: a Schematic of engineered SOX9 and KRT20 loci in dual endogenous reporter lines following GFP and mKate2 (mK2) HDR template integration, respectively. b GSEA of bulk mRNA-seq of mKate2 low /GFP high and mKate2 high /GFP low fractions from HT29 SOX9-mKate2/KRT20-GFP dual reporter line. Two differentiation signatures are indicated in red and green; a normal and aberrant stem cell signature are indicated in orange and blue, respectively. Normalized enrichment scores (NES) and false discovery rates (FDR) for each signature are listed to the right. c Heatmap showing select differentiation and stem cell genes from ( b ). d Ranked log 2 FC plot of CRISPR-Cas9 screen in HT29 SOX9-mKate2/KRT20-GFP line comparing mKate2 low /GFP high and mKate2 high /GFP low fractions. e Beta score of each gene in CRISPR-Cas9 screen targeting epigenetic regulators in HT29 SOX9-mKate2/KRT20-GFP line (3 technical replicates) comparing mKate2 low /GFP high and mKate2 high /GFP low fractions. f Distribution of sgRNA log 2 FC Z-scores of top and bottom hits from CRISPR-Cas9 screen targeting epigenetic regulators. Individual sgRNAs are colored green if enriched and red if depleted in indicated fractions of dual reporter system. g Overlap analysis of shared and exclusive individual sgRNA hits enriched in the 75-100% versus 0-25% GFP+ fractions from the single differentiation reporter system, as well as enriched in mKate2 low /GFP high versus mKate2 high /GFP low fractions or enriched in mKate2 low /GFP high versus mKate2 low /GFP low fractions from the dual reporter system. h Overlap analysis of shared and exclusive individual sgRNA hits depleted in the 75–100% mKate2 versus 0-25% mKate2+ fractions from the single stem reporter system, as well as depleted in mKate2 high /GFP low versus mKate2 low /GFP low fractions, or depleted in mKate2 high /GFP low versus mKate2 low /GFP high fractions from the dual reporter system. i Rank sum of each gene from the CRISPR-Cas9 screen targeting epigenetic regulators in HT29 SOX9-mKate2/ KRT20 -GFP line comparing mKate2 low /GFP high to mKate2 high /GFP low fractions. Boxplots show distribution of sgRNAs per gene. Probability density plots show distribution of enriched (green), depleted (red), and all other sgRNA (gray) rank sums (right panel).

Article Snippet: (Addgene #158560) was used for doxycycline-inducible SOX9 overexpression.

Techniques: CRISPR

Journal: Nature Communications

Article Title: Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

doi: 10.1038/s41467-024-46285-w

Figure Lengend Snippet: a Schematic of gene and protein regulatory network among genetic perturbations that promoted differentiation and impeded aberrant stem-cell activity in the dual reporter system using STRING. b UMAP representation of CRISPR-Cas9 perturbation of select genes coupled with scRNA-seq readout (Perturb-seq). sgRNA assignment of each cell (left). Representation of intestinal differentiation gene signature (right) . c Violin plot depicting the enterocyte gene expression signature in cells with NT control, CTNNB1, SOX9, SMARCB1, and SMARCA4 sgRNAs. P -values determined by Wilcoxon test (* p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001) d GSEA of single cell transcriptomes from cells with CTNNB1, SOX9, SMARCB1, and SMARCA4 sgRNAs relative to NT controls using distinct differentiation and one stem cell signature . e Immunoblot of SMARCB1, KRT20, and GAPDH in HT29 SMARCB1 KD cell lines using 2 shRNAs and HT29 SMARCB1 KO clones using CRISPR-Cas9; clone C1 has 32.9% and clone C2 has 72.6% editing. f . Immunoblot of SMARCB1 and KRT20 (left) and mRNA expression (right) of SMARCB1 , SOX9 , and KRT20 in patient-derived CRC organoid expressing NTC and 2 SMARCB1 shRNAs. g GSEA (top) and heatmap (bottom) of differentiation gene expression signature in bulk RNA-seq data from the patient-derived CRC organoid expressing NTC and 2 SMARCB1 shRNAs. h mRNA expression of SMARCB1 and SOX9 in HT29 SMARCB1 KD cell lines using 2 shRNAs. i mRNA expression of SMARCB1, KRT20, SOX9 in HT115 control and SMARCB1 KD lines engineered to conditionally overexpress SOX9. Source data are provided as a Source Data file.

Article Snippet: (Addgene #158560) was used for doxycycline-inducible SOX9 overexpression.

Techniques: Activity Assay, CRISPR, Gene Expression, Control, Western Blot, Clone Assay, Expressing, Derivative Assay, RNA Sequencing

Journal: Nature Communications

Article Title: Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

doi: 10.1038/s41467-024-46285-w

Figure Lengend Snippet: a Distribution of CTNNB1, SOX9, SMARCB1, KMT2A, and SMARCA4 dependency scores across microsatellite stable (MSS) and microsatellite instability (MSI) CRC cell lines. Dotted red line at a dependency score –1.0. b Relative growth of HT29 CRC cell line expressing NTC or two shRNAs targeting SMARCB1. c Tumor volume of HT29 CRC cell line xenografts expressing NTC shRNA ( n = 6), SMARCB1 sh#1 ( n = 6), or SMARCB1 sh#4 ( n = 6). Tumor volume data shown as mean ± SEM. SMARCB1 immunohistochemistry of representative xenografts at experiment endpoint (right). d Morphology and relative growth of patient-derived CRC organoid upon SMARCB1 knockdown compared to NTC. e Relative growth of patient-derived CRC organoid xenograft expressing NTC shRNA ( n = 7), SMARCB1 sh#1 ( n = 6), or SMARCB1 sh#4 ( n = 8). Relative growth data shown as mean ± SEM. Normalized mRNA expression of SMARCB1 and KRT20 in patient-derived CRC xenografts at experimental endpoint. f Immunohistochemistry of SMARCB1, KRT20, DPP4, LGR5, and SOX9 in patient-derived CRC organoid xenografts expressing NTC or SMARCB1 shRNA. Blue arrows indicate two crypts that escaped SMARCB1 KD. Quantification of SMARCB1 and KRT20 shown in the right panel. g Immunoblot of differentiation markers KRT20 and DPP4, BAF complex members SMARCB1 and SMARCC1, and loading control GAPDH in patient-derived CRC organoid xenografts. Xenografts marked in blue represent paired xenografts grown in the same mouse; L = left and R = right. Source data are provided as a Source Data file.

Article Snippet: (Addgene #158560) was used for doxycycline-inducible SOX9 overexpression.

Techniques: Expressing, shRNA, Immunohistochemistry, Derivative Assay, Knockdown, Western Blot, Control