SQSTM1/p62 CRISPR Activation Plasmid (h): sc-400099-ACT

SQSTM1 encodes sequestosome 1 (p62), a multifunctional adaptor that couples ubiquitinated cargo to selective autophagy via LC3 binding and coordinates proteostasis through interactions with the ubiquitin–proteasome system. p62 is a signaling hub in stress-response pathways, including KEAP1–NRF2 antioxidant signaling and NF-κB activation, and contributes to regulation of apoptosis, inflammation, and metabolic homeostasis. Altered SQSTM1/p62 expression or aggregation is linked to disrupted autophagic flux and proteotoxic stress, features observed across neurodegeneration and other disorders involving chronic cellular stress. As a scaffold integrating degradation and signaling, SQSTM1 is widely studied for its roles in inclusion body formation, mitochondrial quality control, and innate immune regulation.

SQSTM1/p62 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SQSTM1 expression without altering the underlying DNA sequence.

SQSTM1/p62 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SQSTM1 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 SQSTM1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SQSTM1/p62 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SQSTM1 locus and enabling the study of SQSTM1/p62-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SQSTM1/p62 pathway restoration in tumor cells with silenced or reduced SQSTM1 expression.

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