CNPase CRISPR Activation Plasmid (h): sc-402463-ACT

Human CNP encodes 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), an abundant myelin-associated enzyme implicated in oligodendrocyte maturation, myelin sheath maintenance, and cytoskeletal organization. CNPase hydrolyzes 2′,3′-cyclic nucleotides and participates in RNA-associated processes and membrane–cytoskeleton interactions that influence glial process outgrowth and axon–glia coupling. Altered CNP expression or activity has been linked to demyelination phenotypes and neuroinflammatory contexts, making it relevant to studies of white matter integrity and neural connectivity. As a glial-enriched marker, CNPase is frequently used to interrogate pathways governing myelination, stress responses in oligodendrocytes, and mechanisms underlying myelin instability.

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

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

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