Enigma CRISPR Activation Plasmid (h): sc-403254-ACT

Human PDLIM7 encodes Enigma, a PDZ–LIM domain adaptor that localizes to the actin cytoskeleton and focal adhesion–associated structures, linking signaling complexes to cytoskeletal remodeling. Enigma participates in mechanotransduction and cell motility programs by coordinating protein–protein interactions within adhesion and stress fiber networks, influencing pathways that govern cellular architecture and differentiation. Through its scaffolding functions, PDLIM7 has been studied in contexts involving cardiac and skeletal muscle organization, where cytoskeletal integrity and transcriptional regulation are tightly coupled. Dysregulated PDLIM7 expression or localization has been associated in the literature with altered cell migration and tissue remodeling phenotypes relevant to cardiovascular and oncogenic biology.

Enigma CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous PDLIM7 expression without altering the underlying DNA sequence.

Enigma CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the PDLIM7 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 PDLIM7 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Enigma expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native PDLIM7 locus and enabling the study of Enigma-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Enigma pathway restoration in tumor cells with silenced or reduced PDLIM7 expression.

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