CCT A CRISPR Activation Plasmid (h): sc-404021-ACT

PCYT1A encodes choline-phosphate cytidylyltransferase A (CCT A), the rate-limiting enzyme in the CDP-choline (Kennedy) pathway that generates phosphatidylcholine, a major structural lipid of eukaryotic membranes. By controlling phosphatidylcholine synthesis, CCT A influences membrane biogenesis, lipid droplet dynamics, and ER and Golgi homeostasis, linking lipid availability to organelle function and cellular growth. PCYT1A activity interfaces with broader phospholipid remodeling and lipoprotein metabolism, shaping membrane composition and signaling competence. Altered PCYT1A function is associated with inherited disorders of phospholipid metabolism and can impact cellular stress responses and differentiation programs relevant to metabolic and neurodevelopmental phenotypes.

CCT A CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PCYT1A expression without altering the underlying DNA sequence.

CCT A CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PCYT1A 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 PCYT1A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CCT A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PCYT1A locus and enabling the study of CCT A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CCT A pathway restoration in tumor cells with silenced or reduced PCYT1A expression.

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