Hugl-1 CRISPR Activation Plasmid (h): sc-403441-ACT

LLGL1 encodes the human Hugl-1 protein, a conserved regulator of epithelial cell polarity and basolateral membrane organization. Hugl-1 functions within apical–basal polarity networks that coordinate junctional integrity, asymmetric cell division, and cytoskeletal organization, thereby influencing tissue architecture and differentiation. Disruption of polarity control is linked to altered migration and invasive behavior, making LLGL1 a relevant node for studies of epithelial homeostasis, morphogenesis, and tumor-associated loss of polarity. LLGL1 expression and localization are also used to interrogate signaling cross-talk between polarity modules and pathways governing proliferation and cell–cell adhesion.

Hugl-1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous LLGL1 expression without altering the underlying DNA sequence.

Hugl-1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the LLGL1 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 LLGL1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Hugl-1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native LLGL1 locus and enabling the study of Hugl-1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Hugl-1 pathway restoration in tumor cells with silenced or reduced LLGL1 expression.

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