Duffy CRISPR Activation Plasmid (h): sc-406288-ACT

ACKR1 encodes the Duffy antigen/chemokine receptor, an atypical chemokine receptor expressed prominently on erythrocytes and venular endothelial cells that binds inflammatory chemokines including CCL2, CXCL8, and related ligands. By acting as a chemokine sink and transporter, Duffy modulates chemokine gradients, leukocyte trafficking, and inflammatory tone within the vascular microenvironment. Variation in ACKR1 expression is widely used as a model for studying chemokine homeostasis, endothelial biology, and erythrocyte surface antigen regulation, with relevance to inflammation-associated phenotypes and hematologic traits. These functions connect ACKR1 to broader immune signaling networks governing cell migration, cytokine/chemokine signaling, and vascular inflammation.

Duffy CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ACKR1 expression without altering the underlying DNA sequence.

Duffy CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ACKR1 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 ACKR1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Duffy expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ACKR1 locus and enabling the study of Duffy-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Duffy pathway restoration in tumor cells with silenced or reduced ACKR1 expression.

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