Parkin CRISPR Activation Plasmid (m): sc-424514-ACT

Mouse Park2 encodes Parkin, an E3 ubiquitin ligase that coordinates ubiquitin-dependent protein turnover and mitochondrial quality control. Parkin is recruited to damaged mitochondria downstream of PINK1, driving mitophagy through ubiquitination of outer mitochondrial membrane substrates and modulation of autophagosome engagement, with additional roles in proteostasis and stress responses. These activities intersect with pathways governing oxidative stress, bioenergetics, and innate immune signaling linked to mitochondrial dysfunction. Dysregulation of Park2/Parkin function is widely studied in the context of neurodegeneration and cell vulnerability to mitochondrial damage, making it a key target for mechanistic studies of mitochondrial homeostasis.

Parkin CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Park2 expression without altering the underlying DNA sequence.

Parkin CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Park2 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 Park2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Parkin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Park2 locus and enabling the study of Parkin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Parkin pathway restoration in tumor cells with silenced or reduced Park2 expression.

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