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SRP166459
Homo sapiens
26 Downloadable Samples
Illumina HiSeq 2500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T and HepG2 cells were transfected with regular and modified pCAG-BE3-P2A-EGFP or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 21 Downloadable Samples
- Illumina HiSeq 2500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T and HepG2 cells were transfected with pCAG-BE3-P2A-EGFP or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 9 Downloadable Samples
- NextSeq 500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T cells were transfected with pCAG-BE3-P2A-EGFP or variants thereof or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 2 Downloadable Samples
- Illumina HiSeq 2500
Description
Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities. Overall design: HEK293T or HepG2 cells were transfected with P2A-EGFP. Cells were sorted for top 5% GFP based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs.
Publication Title
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Homo sapiens
- 11 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Though p53 mutations are rare in Ewing sarcoma, there is a strong indication that p53-mutant tumors form a particularly bad prognosis group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumor cells containing mutant-p53 in this subset of ES patients.
Publication Title
Variability in functional p53 reactivation by PRIMA-1(Met)/APR-246 in Ewing sarcoma.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 11 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Transient transfection of a Ewing's Sarcoma cell line expressing type I EWS-FLI1 fusion and doxycycline-inducible short hairpin RNA against EWS-FLI1 (A673sh)
Publication Title
Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 10 Downloadable Samples
- Illumina HiSeq 2000
Description
MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3´UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing Sarcoma. miR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be the top EWS-FLI1 activated miRNA. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3´UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signalling. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway Overall design: mRNA profiles of a Ewings Sarcoma cellline (clone of A673 with inducible sh EWS-FLI1 knockdown) treated with microRNA sponges and controls
Publication Title
The role of miR-17-92 in the miRegulatory landscape of Ewing sarcoma.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 16 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulation inhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma.
Sample Metadata Fields
Cell line, Treatment
View Samples- Homo sapiens
- 32 Downloadable Samples
- Illumina HiSeq 2000
Description
Ewing Sarcoma (EwS) is a EWS-FLI1- fusion driven pediatric bone cancer with high metastatic potential. Cellular plasticity, typically regulated via the Rho-pathway, is a prerequisite for metastasis initiation. Here we interrogated the role of the Rho transcriptional effectors MRTFA/B in EwS. We find MRTFB transcriptional function strongly repressed by EWS-FLI1. Under EWS-FLI1-low (knock-down) conditions, MRTFB is activated and antagonizes global EWS-FLI1-dependent transcription. Furthermore, ChIP-Seq revealed strong overlaps in MRTFB and EWS-FLI1 chromatin occupation, especially for EWS-FLI1 suppressed-(anticorrelated) genes. Enrichment of TEAD binding motifs in these shared genomic binding regions, and overlapping transcriptional footprints of MRTFB and TEAD1-4 perturbation led us to propose synergy between MRTFB and TEAD in the regulation of EWS-FLI1 suppressed-anticorrelated genes. Finally, we find F-actin assembly to be already perturbed in our EwS model, F-actin polymerization is perturbed by EWS-FLI1 in our model cell line, however,but pharmacological inhibition of actin polymerization still reduced expression serum-induced expression of MRTFB/YAP-1/TEAD target genes. In summary our data support a model of indirect and direct EWS-FLI1-driven perturbation of MRTFB/YAP-1/TEAD target gene regulation . Overall design: 1. Transient si-RNA mediated knockdown of MRTFA (MKL-1), MRTFB (MKL-2) and doxycyline-induced EWS-FLI1 knockdown in A673/TR/shEF EwS cells (8 samples/replicate: 2 replicates total); 2. Combined transient knockdown of MRTFA, MRTFB and EWS-FLI1 in SK-N-MC EwS cells (4 samples/replicate: 2 replicates total); 3. Combined knockdown of TEAD1-4 by pooling si-RNA against TEAD1, TEAD2, TEAD3 and TEAD 4 combined with doxycycline-inducible EWS-FLI1 knockdown (4 samples/replicate: 8 samples total)
Publication Title
EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulation inhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 16 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Ewing Sarcoma (EwS) is a EWS-FLI1- fusion driven pediatric bone cancer with high metastatic potential. Cellular plasticity, typically regulated via the Rho-pathway, is a prerequisite for metastasis initiation. Here we interrogated the role of the Rho transcriptional effectors MRTFA/B in EwS. We find MRTFB transcriptional function strongly repressed by EWS-FLI1. Under EWS-FLI1-low (knock-down) conditions, MRTFB is activated and antagonizes global EWS-FLI1-dependent transcription. Furthermore, ChIP-Seq revealed strong overlaps in MRTFB and EWS-FLI1 chromatin occupation, especially for EWS-FLI1 suppressed-(anticorrelated) genes. Enrichment of TEAD binding motifs in these shared genomic binding regions, and overlapping transcriptional footprints of MRTFB and TEAD1-4 perturbation led us to propose synergy between MRTFB and TEAD in the regulation of EWS-FLI1 suppressed-anticorrelated genes. Finally, we find F-actin assembly to be already perturbed in our EwS model, F-actin polymerization is perturbed by EWS-FLI1 in our model cell line, however,but pharmacological inhibition of actin polymerization still reduced expression serum-induced expression of MRTFB/YAP-1/TEAD target genes. In summary our data support a model of indirect and direct EWS-FLI1-driven perturbation of MRTFB/YAP-1/TEAD target gene regulation .
Publication Title
EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target gene regulation inhibiting cytoskeletal autoregulatory feedback in Ewing sarcoma.
Sample Metadata Fields
Cell line
View Samples