CTCF CRISPR Activation Plasmid (h): sc-401245-ACT

CTCF (CCCTC-binding factor) is a zinc finger DNA-binding protein that functions as a master chromatin architectural regulator, coordinating enhancer–promoter communication, transcriptional insulation, and long-range genome looping. By shaping topologically associating domains and regulating boundary elements, CTCF influences core processes including gene expression programs, replication timing, and DNA damage responses. Disruption of CTCF-dependent genome organization is linked to widespread transcriptional dysregulation and genomic instability, features frequently studied in the context of developmental disorders and cancer-associated chromatin remodeling. As a central organizer of 3D genome structure, CTCF is widely used in mechanistic studies of epigenetic regulation, lineage specification, and promoter-enhancer control.

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

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

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