αA-crystallin CRISPR Activation Plasmid (h): sc-402047-ACT

CRYAA encodes human αA-crystallin, a small heat shock protein that functions as an ATP-independent molecular chaperone to maintain proteostasis by preventing aggregation of partially unfolded proteins. Highly expressed in the ocular lens, αA-crystallin also contributes to cytoskeletal organization, stress tolerance, and regulation of apoptosis through interactions with client proteins and other chaperone networks. CRYAA activity is linked to cellular responses to oxidative stress and protein quality control pathways that are critical for maintaining lens transparency. Dysregulation or mutation of CRYAA is associated with protein aggregation phenotypes and has been implicated in cataract-related molecular pathology and broader stress-response biology.

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

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

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