MetRS CRISPR Activation Plasmid (h): sc-402763-ACT

Human MARS encodes methionyl‑tRNA synthetase (MetRS), a class I aminoacyl‑tRNA synthetase that ligates methionine to initiator and elongator tRNA^Met, establishing translational initiation and sustaining global protein synthesis. MetRS activity connects amino acid availability to ribosome output and proteostasis, influencing stress responses such as the integrated stress response and mTOR-linked nutrient signaling through its impact on translation capacity. As a core component of cytosolic translation machinery, altered MARS expression or function can perturb cellular growth programs, mitochondrial and ER homeostasis via proteome imbalance, and adaptive responses to amino acid limitation. Dysregulation of translation-related factors, including aminoacyl‑tRNA synthetases, is frequently studied in the context of neurodevelopmental phenotypes, cancer cell fitness, and inflammatory signaling where translational control shapes cell state.

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

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

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