Cdt1 CRISPR Activation Plasmid (h): sc-401014-ACT

Human CDT1 encodes Cdt1, a DNA replication licensing factor that loads the MCM2–7 helicase onto replication origins during late mitosis and G1 to ensure timely S-phase entry. Cdt1 activity is tightly regulated by CDK-dependent phosphorylation, geminin binding, and ubiquitin-mediated proteolysis via CRL4Cdt2 and SCFSkp2 pathways to prevent rereplication and preserve genome stability. Dysregulation of CDT1 can drive replication stress, aberrant origin firing, and DNA damage signaling, processes frequently linked to chromosomal instability in cancer and other proliferative disorders. As a core component of the origin licensing network, Cdt1 is widely studied in cell-cycle control, checkpoint responses, and replication-coupled DNA repair.

Cdt1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CDT1 expression without altering the underlying DNA sequence.

Cdt1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CDT1 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 CDT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Cdt1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CDT1 locus and enabling the study of Cdt1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Cdt1 pathway restoration in tumor cells with silenced or reduced CDT1 expression.

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