GW182 CRISPR Activation Plasmid (h): sc-400906-ACT

Human TNRC6A encodes GW182, a core scaffold protein of GW/P-bodies that couples Argonaute-bound microRNAs to post-transcriptional gene silencing. GW182 interacts with CCR4–NOT and PAN2–PAN3 deadenylase complexes to promote mRNA deadenylation, decapping, and decay, and it also contributes to translational repression of target transcripts. Through these activities, TNRC6A helps shape gene expression programs involved in cell-cycle control, differentiation, and stress responses. Dysregulation of miRNA-mediated silencing and P-body dynamics has been linked to oncogenic signaling, neurodevelopmental phenotypes, and immune-related processes, making TNRC6A a useful node for mechanistic studies of RNA regulation.

GW182 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TNRC6A expression without altering the underlying DNA sequence.

GW182 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TNRC6A 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 TNRC6A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous GW182 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TNRC6A locus and enabling the study of GW182-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of GW182 pathway restoration in tumor cells with silenced or reduced TNRC6A expression.

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