GAK CRISPR Activation Plasmid (h): sc-404235-ACT

Human GAK (cyclin G–associated kinase) is a serine/threonine kinase that coordinates clathrin-mediated endocytosis by regulating AP2 adaptor and clathrin coat dynamics, thereby influencing receptor internalization and trafficking. Through control of vesicle uncoating and endosomal sorting, GAK impacts signaling outputs from membrane receptors and contributes to maintenance of cellular proteostasis and membrane homeostasis. Its activity intersects with cell cycle–linked regulatory networks and stress-responsive pathways that depend on efficient vesicular transport. Altered GAK expression or function has been associated in the literature with neurodegeneration- and oncology-relevant phenotypes, supporting its use as a mechanistic node for studying pathway remodeling in disease contexts.

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

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

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