HLA-E CRISPR Activation Plasmid (h): sc-403124-ACT

HLA-E encodes a non-classical MHC class I molecule that presents a restricted peptide repertoire, including leader peptides from other HLA class I proteins, to regulate immune surveillance. At the cell surface, HLA-E primarily engages inhibitory and activating receptors such as CD94/NKG2A and CD94/NKG2C on NK cells and subsets of T cells, integrating into antigen presentation and immune checkpoint-like signaling that tunes cytotoxic responses. By shaping NK- and T-cell activation thresholds, HLA-E influences responses to infection, inflammation, and tumor–immune interactions, and altered expression has been associated with immune evasion phenotypes in multiple disease contexts. Its regulation intersects with cytokine-driven pathways, including interferon signaling, that modulate antigen-processing and presentation programs.

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

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

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