PIG-A CRISPR Activation Plasmid (m): sc-422226-ACT

Mouse Piga encodes PIG-A, an essential catalytic subunit of the glycosylphosphatidylinositol (GPI)-anchor biosynthesis machinery that initiates the first step of GPI precursor assembly in the endoplasmic reticulum. By enabling GPI anchoring, PIG-A supports surface expression and membrane localization of numerous proteins involved in signal transduction, adhesion, enzymatic activity, and immune regulation. Perturbation of PIG-A function disrupts GPI-anchor biogenesis, altering membrane protein trafficking and cell-surface proteome composition, and is widely used to study GPI-anchor–dependent pathways in hematopoietic and other lineages. Dysregulated GPI-anchor biosynthesis is implicated in disease biology, including clonal hematologic disorders, providing a mechanistic framework for modeling membrane-anchored protein deficits in experimental systems.

PIG-A CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Piga expression without altering the underlying DNA sequence.

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

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