CRY2 CRISPR Activation Plasmid (h): sc-403171-ACT

Human CRY2 encodes cryptochrome 2, a blue-light–responsive flavoprotein that functions as a core repressor within the circadian transcriptional–translational feedback loop. CRY2 regulates CLOCK:BMAL1-driven gene expression by interacting with PER proteins and modulating ubiquitin-dependent turnover, thereby shaping daily rhythms in metabolism, cell cycle timing, and hormone signaling. Through coupling to post-translational modification networks, including phosphorylation and E3 ligase–mediated proteostasis, CRY2 influences transcriptional programs across peripheral tissues. Dysregulated CRY2 activity or expression has been associated with altered sleep–wake regulation and circadian misalignment linked to metabolic and neuropsychiatric phenotypes, making it a useful node for pathway-centric studies.

CRY2 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous CRY2 expression without altering the underlying DNA sequence.

CRY2 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the CRY2 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 CRY2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous CRY2 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native CRY2 locus and enabling the study of CRY2-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of CRY2 pathway restoration in tumor cells with silenced or reduced CRY2 expression.

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