CPI-17 CRISPR Activation Plasmid (h): sc-401966-ACT

Human PPP1R14A encodes CPI-17, a phosphorylation-dependent inhibitor of myosin light chain phosphatase (MLCP) that amplifies smooth muscle contraction by sustaining myosin regulatory light chain phosphorylation. Upon activation by kinases such as PKC and ROCK, CPI-17 links G protein–coupled receptor signaling to Ca²⁺ sensitization, integrating pathways that control cytoskeletal dynamics, cell contractility, and motility. This regulatory node is broadly relevant to vascular and airway smooth muscle physiology and to mechanisms of actomyosin remodeling in non-muscle cells. Dysregulated PPP1R14A/CPI-17 signaling has been investigated in contexts involving abnormal contractile tone and remodeling, including cardiovascular and pulmonary disease biology.

CPI-17 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PPP1R14A expression without altering the underlying DNA sequence.

CPI-17 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PPP1R14A 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 PPP1R14A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CPI-17 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PPP1R14A locus and enabling the study of CPI-17-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CPI-17 pathway restoration in tumor cells with silenced or reduced PPP1R14A expression.

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