HLA-C CRISPR Activation Plasmid (h): sc-401517-ACT

HLA-C encodes a classical MHC class I heavy chain that forms a heterodimer with β2-microglobulin to present endogenously processed peptides at the cell surface for CD8+ T cell surveillance. As part of antigen processing and presentation, HLA-C integrates inputs from proteasomal peptide generation, TAP-dependent transport into the endoplasmic reticulum, and peptide loading to shape immunopeptidome composition. HLA-C also serves as a key ligand for inhibitory and activating killer cell immunoglobulin-like receptors (KIRs) on natural killer cells, influencing immune recognition thresholds. Allelic variation and altered expression of HLA-C are widely studied in the context of infection, inflammatory and autoimmune processes, cancer immune interactions, and transplant compatibility.

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

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

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