CENP-B CRISPR Activation Plasmid (h): sc-401356-ACT

CENPB encodes centromere protein B (CENP-B), a sequence-specific DNA-binding factor that recognizes α-satellite centromeric repeats and contributes to centromere organization and kinetochore assembly. Through interactions with centromeric chromatin and the CENP-A–containing nucleosome environment, CENP-B supports chromosome segregation fidelity and mitotic progression. Altered centromere function and chromosomal instability are common features of tumor evolution, making CENP-B regulation relevant for studies of aneuploidy, cell cycle checkpoints, and genome maintenance. CENP-B is also a major autoantigen targeted by anti-centromere antibodies, linking CENPB biology to investigations of centromere-directed autoimmunity and nuclear antigen processing.

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

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

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