HLA-B CRISPR Activation Plasmid (h): sc-400627-ACT

HLA-B encodes a polymorphic MHC class I heavy chain that pairs with β2-microglobulin to present intracellularly derived peptides to CD8+ T cells, shaping immune surveillance and cytotoxic responses. Its expression is tightly regulated by interferon-stimulated signaling and antigen processing and presentation pathways, including proteasomal peptide generation and TAP-mediated peptide transport into the endoplasmic reticulum. Variation in HLA-B influences the peptide repertoire and modulates allorecognition and host–pathogen interactions, linking this locus to immune-mediated disease susceptibility and transplant compatibility. Altered HLA-B expression or allele usage is also relevant to studies of immune evasion and inflammatory microenvironments where antigen presentation is dynamically remodeled.

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

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

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