grna empty vector  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: A modular dCas9-based recruitment platform for combinatorial epigenome editing

doi: 10.1093/nar/gkad1108

Figure Lengend Snippet: Recruitment of DNMT3A, KRAB and EZH2 to induce targeted transcriptional repression via the SSSavi system. RT-qPCR quantification of target gene transcriptional repression induced by transient transfection of SSSavi plasmids in HSB6G (dCas9-SSSavi and 6x sgRNA) stable cells. ( A ) Catchers were tested individually or in four-way combinations for each of three effectors: DNMT3A (D3A), EZH2 (E), and/or KRAB (K) fused to either SpyCatcher, SnoopCatcher, α GCN4 or Traptavidin, across four different target genes ( PACC1 , B2M , RBM3 , and HINT1 ). Fold change in expression was calculated compared to transfection with α GCN4-mCherry alone (baseline control, black dotted lines). *Independent sample t-tests with Benjamini–Hochberg correction comparing effector to α GCN4-mCherry, where P -values < 0.05. ( B ) A schematic of the six possible three-way combinations indicating the order in which effectors (D3A, K, and E) were recruited onto the SSSavi docking array due to their differential fusion to SpyCatcher, α GCN4 or Traptavidin. ( C ) RT-qPCR quantification of target gene transcript abundance following transient transfection with all possible three-way combinations of spatial ordering of D3A, K and E recruited to the SSSavi docking array. Two control samples were also included: α GCN4-mCherry as the non-catalytic baseline control (black dotted line) and SpyCatcher fused to D3A as a single epimodifier positive control. Bars with different letters indicate a significant difference as calculated by independent sample t -tests. a: compares D3A-K-E to aGCN4-mCherry, and b: compares D3A-K-E to E-K-D3A ( P -values < 0.05). ( A , C ) Bar graphs indicate sample mean ( n = 5, biological replicates, log 2 fold change calculated compared to α GCN4-mCherry control samples, sorted for GFP).

Article Snippet: The control sample, α GCN4-mCherry, was cloned by PCR amplifying both the mCherry from LLP469 pEF1a-mCherry-EMPTY-gRNA (Addgene, #100958) and the backbone of α GCN4-p65HSF1-CO, followed by Gibson assembly, replacing the p65HSF1 domain with mCherry.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Control, Positive Control

Journal: Nucleic Acids Research

Article Title: A modular dCas9-based recruitment platform for combinatorial epigenome editing

doi: 10.1093/nar/gkad1108

Figure Lengend Snippet: Downregulation of EPCAM mRNA and protein levels in HepG2 liver cancer cells by the SSSavi system. ( A ) RT-qPCR quantification of target gene transcriptional repression induced by transient transfection of SSSavi catcher plasmids in the HepSB (HepG2 cells with dCas9-SSSavi and BirA stable) cell line using DNMT3A (D3A), EZH2 (E), and KRAB (K), as well as a 6× sgRNA plasmid. Fold change in expression is calculated compared to transfection with α GCN4-mCherry alone (baseline control, black dotted lines). Bar graphs indicate sample mean with different letters representing different P -values as calculated by independent sample t -tests with Benjamini–Hochberg correction, comparing D3A-K-E to α GCN4-mCherry (a: P = 4.5 × 10 −3 ( EPCAM ), 8.8 × 10 −3 ( HINT1 ), b: P = 0.1 ( B2M )). n = 4, biological replicates, log 2 fold change calculated compared to α GCN4-mCherry control samples, sorted for GFP (catchers) and mCherry (6x sgRNA)). ( B ) A schematic of the different effectors recruited onto the SSSavi docking array, highlighting the different spatial ordering. ( C ) Flow cytometry analysis of HepSB cells immunostained with EPCAM antibody, comparing the average percentage of positive cells (and error variance for two replicates) across four samples transfected with α GCN4-mCherry alone (grey), Spy-D3A alone (green), K-E-D3A (blue) or D3A-K-E (red) effector combinations.

Article Snippet: The control sample, α GCN4-mCherry, was cloned by PCR amplifying both the mCherry from LLP469 pEF1a-mCherry-EMPTY-gRNA (Addgene, #100958) and the backbone of α GCN4-p65HSF1-CO, followed by Gibson assembly, replacing the p65HSF1 domain with mCherry.

Techniques: Quantitative RT-PCR, Transfection, Stable Transfection, Plasmid Preparation, Expressing, Control, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: A modular dCas9-based recruitment platform for combinatorial epigenome editing

doi: 10.1093/nar/gkad1108

Figure Lengend Snippet: Effect of dCas9-SSSavi D3A-K-E upon histone and DNA methylation. ( A ) Box and whisker plots depicting DNA methylation levels at individual CpG sites in samples transfected with D3A-K-E ( n = 2) or α GCN4-mCherry ( n = 1) as measured by WGBS and quantified at all CpG sites located in the ±500 bp region centred on the sgRNA binding site (number of CpG sites for each target gene: KL 76, EPCAM 52, PACC1 46, B2M 38, RBM3 16 and HINT1 30). ( B ) H3K4me3 ChIP-seq cumulative read counts (SES normalised) for the six target genes (FDR *<0.15 or **<0.1). ( C ) Genome browser display of the B2M promoter and sgRNA binding site (highlighted in grey). Sets of experiments include (from top to bottom): dCas9 ChIP-seq coverage, WGBS (mCG/CG) and H3K4me3, H3K9me3 and H3K27me3 ChIP-seq coverage (comparing D3A-K-E to α GCN4-mCherry replicate samples, sorted for GFP positive cells).

Article Snippet: The control sample, α GCN4-mCherry, was cloned by PCR amplifying both the mCherry from LLP469 pEF1a-mCherry-EMPTY-gRNA (Addgene, #100958) and the backbone of α GCN4-p65HSF1-CO, followed by Gibson assembly, replacing the p65HSF1 domain with mCherry.

Techniques: DNA Methylation Assay, Whisker Assay, Transfection, Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: A modular dCas9-based recruitment platform for combinatorial epigenome editing

doi: 10.1093/nar/gkad1108

Figure Lengend Snippet: Transcriptional activation and repression of 15 target genes. HSB cells (HEK293T dCas9-SSSavi and BirA stable) transfected with a 15× multiplexed sgRNA array and either ( A ) all four catchers fused to p65HSF1 or ( B ) the strongest three-way combination of repressor domains, D3A-K-E ( n = 5, biological replicates, bar graphs indicate sample mean, log 2 fold change calculated compared to α GCN4-mCherry control samples, sorted for GFP, TPM: transcripts per million). Independent sample t -tests comparing effector to α GCN4-mCherry, where P -values *<0.01, **<0.001, ***<0.0001.

Article Snippet: The control sample, α GCN4-mCherry, was cloned by PCR amplifying both the mCherry from LLP469 pEF1a-mCherry-EMPTY-gRNA (Addgene, #100958) and the backbone of α GCN4-p65HSF1-CO, followed by Gibson assembly, replacing the p65HSF1 domain with mCherry.

Techniques: Activation Assay, Transfection, Control