NUAK1/ARK5 CRISPR Activation Plasmid (h): sc-403053-ACT

NUAK1 (also known as ARK5) encodes a serine/threonine kinase in the AMPK-related kinase family that couples energy and stress cues to cellular survival programs. NUAK1/ARK5 participates in signaling networks downstream of LKB1 and intersects with pathways controlling metabolism, cytoskeletal dynamics, adhesion, and cell-cycle progression, including modulation of MYPT1/PP1 signaling and actomyosin contractility. Through these functions, NUAK1 has been implicated in regulation of proliferation, migration, and stress tolerance in multiple cell types. Dysregulated NUAK1 activity and expression have been associated with tumor biology and other contexts where metabolic adaptation and invasive behavior are under investigation.

NUAK1/ARK5 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NUAK1 expression without altering the underlying DNA sequence.

NUAK1/ARK5 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NUAK1 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 NUAK1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous NUAK1/ARK5 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NUAK1 locus and enabling the study of NUAK1/ARK5-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of NUAK1/ARK5 pathway restoration in tumor cells with silenced or reduced NUAK1 expression.

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