PIP5KIII CRISPR Activation Plasmid (h): sc-402813-ACT

PIKFYVE encodes phosphatidylinositol-3-phosphate 5-kinase type III (PIP5KIII), a lipid kinase that generates PI(3,5)P2 and supports endolysosomal membrane identity, vesicle trafficking, and lysosome homeostasis. PIP5KIII functions within the PIKFYVE–VAC14–FIG4 complex to regulate endosome maturation, autophagic flux, and nutrient-responsive signaling, linking phosphoinositide metabolism to cellular stress adaptation. Disruption of this pathway alters vacuole/lysosome dynamics and is associated with neurodegenerative and neuromuscular phenotypes, making PIKFYVE a key node for studying membrane trafficking defects. Human PIKFYVE regulation is also relevant to investigations of pathogen entry, receptor turnover, and metabolic signaling that depend on endosomal sorting.

PIP5KIII CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PIKFYVE expression without altering the underlying DNA sequence.

PIP5KIII CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PIKFYVE 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 PIKFYVE transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PIP5KIII expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PIKFYVE locus and enabling the study of PIP5KIII-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PIP5KIII pathway restoration in tumor cells with silenced or reduced PIKFYVE expression.

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