MANEAL CRISPR Activation Plasmid (h): sc-414652-ACT

MANEAL encodes a poorly characterized membrane-associated protein that is predicted to localize to endomembrane compartments and may contribute to intracellular trafficking and protein handling within the secretory pathway. Emerging annotation and domain features suggest potential links to ER–Golgi transport or membrane organization processes that influence proteostasis and cellular stress responses. As a result, MANEAL is of interest for studying how endomembrane dynamics shape cell-state regulation, including proliferation and differentiation programs. Altered expression patterns reported across genomic and transcriptomic datasets motivate investigation of MANEAL as a context-dependent modulator in disease-relevant cellular phenotypes, particularly in models where secretory pathway integrity is perturbed.

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

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

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