PAPD1 CRISPR Activation Plasmid (h): sc-412459-ACT

MTPAP encodes the mitochondrial poly(A) polymerase PAPD1, an RNA-processing enzyme that adds poly(A) tails to mitochondrial mRNAs to support transcript stability, maturation, and efficient mitochondrial translation. Through its role in mitochondrial gene expression, PAPD1 contributes to oxidative phosphorylation capacity, respiratory chain homeostasis, and broader mitochondrial quality control programs. Dysregulated mitochondrial RNA polyadenylation can perturb energy metabolism and amplify cellular stress responses linked to neuromuscular and neurodegenerative phenotypes. As a result, MTPAP/PAPD1 is commonly studied in pathways connecting mitochondrial RNA biology to bioenergetics, proteostasis, and cell survival under metabolic stress.

PAPD1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MTPAP expression without altering the underlying DNA sequence.

PAPD1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MTPAP 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 MTPAP transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PAPD1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MTPAP locus and enabling the study of PAPD1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PAPD1 pathway restoration in tumor cells with silenced or reduced MTPAP expression.

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