Cystinosin CRISPR Activation Plasmid (h): sc-405040-ACT

CTNS encodes cystinosin, a lysosomal membrane transporter that mediates cystine efflux from the lysosomal lumen to the cytosol, supporting intracellular redox balance and amino acid homeostasis. By regulating lysosomal cystine clearance, cystinosin links lysosome function to broader metabolic processes including autophagy-lysosome dynamics and cellular stress responses. Altered CTNS activity perturbs lysosomal storage and downstream proteostasis pathways, making it a key node for studying organelle quality control and metabolic adaptation. CTNS is implicated in cystinosis-related lysosomal dysfunction and is frequently examined in models of renal proximal tubule physiology and systemic amino acid transport.

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

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

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