TTC35 CRISPR Activation Plasmid (h): sc-405724-ACT

EMC2 encodes TTC35, a conserved tetratricopeptide repeat–containing component of the endoplasmic reticulum membrane protein complex (EMC). This complex supports insertion and maturation of multipass membrane proteins, contributing to ER proteostasis, membrane trafficking, and cellular stress responses. Disruption of EMC subunits can impair biogenesis of receptors, channels, and transporters, linking EMC2/TTC35-regulated processes to altered secretory pathway function and sensitivity to ER stress. As a result, EMC2 is frequently studied in the context of protein quality control networks and disease-relevant phenotypes driven by defective membrane protein folding or trafficking.

TTC35 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EMC2 expression without altering the underlying DNA sequence.

TTC35 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EMC2 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 EMC2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous TTC35 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EMC2 locus and enabling the study of TTC35-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of TTC35 pathway restoration in tumor cells with silenced or reduced EMC2 expression.

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