Clock CRISPR Activation Plasmid (h): sc-401261-ACT

Human CLOCK encodes a basic helix–loop–helix PAS-domain transcription factor that forms a heterodimer with ARNTL/BMAL1 to drive circadian gene expression through E-box regulatory elements. This core oscillator regulates rhythmic transcription of pathways governing metabolism, cell cycle progression, DNA damage responses, and neuroendocrine signaling, linking time-of-day cues to cellular homeostasis. CLOCK-dependent programs influence chromatin state and transcriptional output across tissues, shaping coordinated physiological rhythms. Dysregulated CLOCK activity and circadian misalignment have been associated with sleep and mood phenotypes, metabolic dysfunction, and altered proliferative states relevant to complex disease biology.

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

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

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