COL2A1 CRISPR Activation Plasmid (h): sc-400359-ACT

COL2A1 encodes the α1 chain of type II collagen, a major fibrillar component of cartilage extracellular matrix that supports tissue tensile strength and hydration through interactions with proteoglycans. Its expression is tightly linked to chondrocyte differentiation and matrix assembly programs coordinated by SOX9-driven transcriptional networks and extracellular matrix remodeling pathways. Perturbation of COL2A1 alters cartilage structural integrity and can disrupt endochondral development processes relevant to skeletal and joint biology. Genetic variation or dysregulated expression of COL2A1 has been associated with heritable cartilage and skeletal dysplasias, making it a frequent target for studying extracellular matrix homeostasis and chondrocyte lineage regulation.

COL2A1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous COL2A1 expression without altering the underlying DNA sequence.

COL2A1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the COL2A1 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 COL2A1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous COL2A1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native COL2A1 locus and enabling the study of COL2A1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of COL2A1 pathway restoration in tumor cells with silenced or reduced COL2A1 expression.

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