C-Nap1 CRISPR Activation Plasmid (h): sc-403009-ACT

Human CEP250 encodes C-Nap1, a centrosomal coiled-coil protein concentrated at the proximal ends of centrioles where it helps maintain centrosome cohesion and organizes microtubule anchoring. C-Nap1 participates in centrosome separation during cell cycle progression through phosphorylation-dependent remodeling, supporting accurate spindle assembly, chromosome segregation, and ciliogenesis-linked processes. Disruption of centrosome integrity and microtubule organization is broadly relevant to chromosomal instability phenotypes and has been associated with neurodevelopmental and retinal disease contexts where centrosome and cilia function are critical. As a result, CEP250 is routinely studied in pathways governing centrosome dynamics, mitotic fidelity, and cytoskeletal organization.

C-Nap1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CEP250 expression without altering the underlying DNA sequence.

C-Nap1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CEP250 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 CEP250 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous C-Nap1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CEP250 locus and enabling the study of C-Nap1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of C-Nap1 pathway restoration in tumor cells with silenced or reduced CEP250 expression.

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