EWS CRISPR Activation Plasmid (h): sc-401036-ACT

EWSR1 encodes the RNA-binding protein EWS, a member of the FET family that couples transcriptional regulation with RNA processing. EWS participates in co-transcriptional splicing, mRNA transport, and stress granule dynamics, and it interfaces with RNA polymerase II–dependent transcription programs to shape gene expression. Chromosomal rearrangements involving EWSR1 generate oncogenic fusion proteins that rewire transcriptional networks and are central to the biology of Ewing sarcoma and related soft tissue tumors. Altered EWSR1 function is also linked to broader defects in genome stability and RNA metabolism that are relevant to mechanistic studies of cellular transformation and differentiation.

EWS CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EWSR1 expression without altering the underlying DNA sequence.

EWS CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EWSR1 locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the EWSR1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EWS expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EWSR1 locus and enabling the study of EWS-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EWS pathway restoration in tumor cells with silenced or reduced EWSR1 expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.