sgrna scaffold  (Addgene inc)

Journal: bioRxiv

Article Title: Marker-based CRISPR screening reveals a MED12-p63 interaction that activates basal identity in pancreatic ductal adenocarcinoma

doi: 10.1101/2023.10.24.563848

Figure Lengend Snippet: a, Diagram illustrating the workflow of KRT5 or p63 genome-wide reporter screens. b, Flow cytometry staining profiles for CRISPRi-mediated TP63 or KRT5 knockdown in KLM1 cells. Secondary staining with AF647-conjugated anti-rabbit antibody (area-filled curves) or unstained controls (outline-only curves) show the signal distribution of p63-(left) or KRT5-(right) stained cells upon gene knockdown. A minimum of 10,000 events were collected and plotted for each sample. c, Metaplot of p63- and KRT5-based reporter genome-wide CRISPRi screen results in 3 independent cell lines (KLM1, T3M4 and BxPC3). Average β scores of p63 and KRT5 screens are plotted such that each dot represents one promoter-defined gene. The size of each dot is proportional to the inverse of the standard deviation of the β values across cell lines. Important mammalian general transcriptional regulators are highlighted according to the legend. β scores were calculated using MAGeCK with the MLE option, with negative β scores denoting enrichment in the marker low population. d, Scatterplot of the average β scores in the p63 (top) or KRT5 (bottom) reporter screens across KLM1, T3M4, and BxPC3, and the median CERES cancer cell line essentiality score from the Cancer Dependency Map. e, Scatterplot of p63-based reporter screens β scores using genome-wide CRISPRi or CRISPR knockout libraries in KLM1 cells. Genes belonging to the Mediator complex are highlighted in red. f, Western blot of whole cell lysates at day 6 post-infection with lentiviral CRISPR knockout sgRNA targeting MED12 or negative control sgRNAs in T3M4 and KLM1 cells.

Article Snippet: The sgRNA lentiviral expression vector with optimized sgRNA scaffold backbone (LRG2.1T-puromycin, Addgene, #125594) was used for CRISPR knockout, interference and activation (a list of sgRNAs used in this study is provided in Supplementary Table 5).

Techniques: Genome Wide, Flow Cytometry, Staining, Knockdown, Standard Deviation, Marker, CRISPR, Knock-Out, Western Blot, Infection, Negative Control

Journal: bioRxiv

Article Title: Marker-based CRISPR screening reveals a MED12-p63 interaction that activates basal identity in pancreatic ductal adenocarcinoma

doi: 10.1101/2023.10.24.563848

Figure Lengend Snippet: a, Heatmap of z-scored variance-stabilizing transformed gene counts of top 250 overexpressed and downregulated genes upon TP63 knockout in T3M4 cells. Non-targeting sgRNAs or knockout of ROSA26 (negative controls); CDK1 , PCNA , or RPA3 (lethal controls); TP63 ; or MED12 were performed with two to four different sgRNAs and three biological replicates each. Each column corresponds to an individual RNA-seq sample, and each row displays the z-score of variance-stabilized transformed counts (calculated with DESeq2) across samples. Samples are clustered by Euclidean distance according to the dendrogram on top of the figure. Genes associated with basal (right) and classical (left) PDAC are labeled on the side. b, Representative GSEA plots of T3M4 MED12 knockout using gene signatures derived from human basal PDAC tumors (Chan-Seng-Yue et al. 2020) and direct p63 gene targets in PDAC (Somerville et al. 2018). The plots were generated and the normalized enrichment scores (NES) and statistics were calculated using DESeq2-derived log2FC values of MED12 vs ROSA26 KO in GSEApy. Three biological replicates were used for each sample. Complete GSEA analysis for all the sgRNA tested can be found in Supplementary Table 2. c, Time-course RT-qPCR of TP63 and its target gene KRT5 after lentiviral infection with CRISPRi sgRNA targeting TP63 (2 sgRNA), MED12 (3 sgRNA), non-targeting sgRNAs (2 sgRNA), or uninfected control T3M4 cells. -ΔΔCt values are plotted as the average of each sgRNA normalized to the average of housekeeping genes ACTB and B2M . For each gene perturbation, the average -ΔΔCt value is shown in a solid line, and the 95% confidence intervals are shown as translucid intervals. MED12 knockdown inflection point (∼ day 5) is marked by a vertical black dashed line. d, RT-qPCR of TP63 and its target genes S100A2 , IL1B and KRT6A after TP63 or empty vector cDNA overexpression and MED12 (2 sgRNA) or non-targeting (2 sgRNA) CRISPRi sgRNAs. RNA was collected at day 5.5 post-infection with lentiviral sgRNAs, and three days after cDNA overexpression. mRNA fold change values are calculated as 2 -ΔΔCt normalized to the average of housekeeping genes ACTB , B2M and PPIA . *: double-sided t-test p-value<0.05. e, Metaplots and heatmaps of p63, MED12, and H3K27ac chromatin occupancy in KLM1 cells. Signal intensity values are centered around p63 peaks annotated by MACS2 and sorted by p63 intensity. f, Metaplots of p63 (left) and MED12 (right) genome occupancy signals centered around MED12 peaks at basal enhancers in KLM1 cells upon ROSA26 or TP63 knockout (2 sgRNA). g, ChIP-seq tracks of p63, MED12, and H3K27ac normalized occupancy at select basal-specific p63 direct target loci in KLM1 cells upon ROSA26 or TP63 knockout. Normalized enrichment values were generated with DeepTools bamCoverage -RPCG. h, Metaplots of p63 (left) and MED12 (right) genome occupancy signals centered around MED12-occupied peaks surrounding p63 direct target genes in SUIT2 CRISPRa- TP63 lines. e,f,h, Metaplots and heatmaps were generated using DeepTools computeMatrix and plotHeatmap.

Article Snippet: The sgRNA lentiviral expression vector with optimized sgRNA scaffold backbone (LRG2.1T-puromycin, Addgene, #125594) was used for CRISPR knockout, interference and activation (a list of sgRNAs used in this study is provided in Supplementary Table 5).

Techniques: Transformation Assay, Knock-Out, RNA Sequencing, Labeling, Derivative Assay, Generated, Quantitative RT-PCR, Infection, Control, Knockdown, Plasmid Preparation, Over Expression, ChIP-sequencing

Journal: bioRxiv

Article Title: Marker-based CRISPR screening reveals a MED12-p63 interaction that activates basal identity in pancreatic ductal adenocarcinoma

doi: 10.1101/2023.10.24.563848

Figure Lengend Snippet: a, FLAG immunoprecipitation of transiently transfected p63-C-3xFLAG in HEK293T nuclear lysates. Ponceau staining and western blot of FLAG and endogenous MKM (MED12) and core Mediator (MED23) subunits are shown for input and IP samples. The two bands in the Ponceau under p63 correspond to denatured IgG fragments. b, FLAG immunoprecipitation of transiently transfected N-3xFLAG-p63 or p63-C-3xFLAG in HEK293T nuclear lysates in the presence or absence of ethidium bromide (EtBr, final concentration of 50μg/mL). Ponceau and western blot of endogenous MKM (MED12, CDK8) and core Mediator (MED1) subunits are shown for input and IP samples. c, MBP-pulldown of purified MBP or MBP-p63 incubated with nuclear lysates of endogenously tagged 3NHA-CDK8 HeLa cells. Nuclear lysate was equally split between MBP and MBP-p63 for pulldown. Ponceau (left) shows the immobilized MBP fusion proteins, and HA stains the 3xHA-tagged CDK8. Only pulldown results are shown, as the signal was undetectable in the 1% input. d, Western blot of MBP-pulldown of purified MBP-p63 incubated with Sf9 cell lysates expressing different individual human Mediator subunits. 1% input is shown in the left lane. e, MBP pulldown of MBP or MBP-p63 incubated with human MED12-expressing Sf9 lysates. f, Glycerol gradient sedimentation of purified MBP or MBP-p63 incubated with human 4-subunit MKM. Glycerol percentages are indicated above the blots; larger complexes will migrate farther down the gradient. Black dashed boxes show glycerol gradient eluted fractions of individual p63, MBP, or MKM, as well as MBP-MKM incubation samples which did not display shifted migration pattern. Red dashed boxes highlight p63-MKM glycerol gradients that eluted in size-shifted fractions. g, Scatterplot depicting gene expression changes upon MED12 or MED13/MED13L knockout. DESeq2-derived log2FC for all expressed genes are plotted. Representative plot of 2 different sgRNA per gene. Select basal genes are highlighted. h, GSEA plots of MED13 / MED13L double knockout using gene signatures derived from human basal PDAC tumors (Chan-Seng-Yue et al. 2020, left) and direct p63 gene targets in PDAC (Somerville et al. 2018, right). Complete GSEA analysis for all the sgRNA tested can be found in Supplementary Table 2.

Article Snippet: The sgRNA lentiviral expression vector with optimized sgRNA scaffold backbone (LRG2.1T-puromycin, Addgene, #125594) was used for CRISPR knockout, interference and activation (a list of sgRNAs used in this study is provided in Supplementary Table 5).

Techniques: Immunoprecipitation, Transfection, Staining, Western Blot, Concentration Assay, Purification, Incubation, Expressing, Sedimentation, Migration, Gene Expression, Knock-Out, Derivative Assay, Double Knockout