HEATR5B CRISPR Activation Plasmid (h2): sc-412293-ACT-2

Human HEATR5B encodes a HEAT repeat–containing protein predicted to function as a scaffolding factor that organizes multiprotein assemblies and supports intracellular trafficking and cytoskeletal-linked processes. Based on the known roles of HEAT-repeat proteins, HEATR5B is expected to influence nucleocytoplasmic transport, protein complex dynamics, and stress-responsive regulation of gene expression, with downstream effects on cell growth and differentiation pathways. Altered HEATR5B activity or expression may contribute to pathogenic mechanisms involving disrupted transport homeostasis and aberrant signaling, making it relevant to studies of complex cellular dysfunction in human disease. Gene editing of HEATR5B is useful for generating knockout or tagged cell models to map interactomes, define subcellular localization, and interrogate pathway-level phenotypes using transcriptomic and proteomic readouts.

HEATR5B CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous HEATR5B expression without altering the underlying DNA sequence.

HEATR5B CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the HEATR5B 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 HEATR5B transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HEATR5B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native HEATR5B locus and enabling the study of HEATR5B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HEATR5B pathway restoration in tumor cells with silenced or reduced HEATR5B expression.

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