Cdt2 CRISPR Activation Plasmid (h): sc-407212-ACT

Human DTL encodes Cdt2, a substrate receptor for the CRL4^Cdt2 E3 ubiquitin ligase complex that coordinates cell-cycle progression with DNA replication and repair. Cdt2 promotes S-phase fidelity by targeting key replication licensing and checkpoint factors, including CDT1, p21 (CDKN1A), and SETD8, for PCNA-dependent ubiquitination and proteasomal degradation during DNA synthesis and after genotoxic stress. Through these functions it influences replication origin control, genome stability, and DNA damage response signaling. Dysregulated DTL/Cdt2 activity has been associated with proliferative phenotypes and altered checkpoint control in cancer-relevant contexts, making it a useful node for studying oncogenic replication stress and cell-cycle vulnerabilities.

Cdt2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous DTL expression without altering the underlying DNA sequence.

Cdt2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the DTL 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 DTL transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Cdt2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native DTL locus and enabling the study of Cdt2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Cdt2 pathway restoration in tumor cells with silenced or reduced DTL expression.

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