GlyRS CRISPR Activation Plasmid (h): sc-404105-ACT

GARS encodes human glycyl‑tRNA synthetase (GlyRS), a cytosolic aminoacyl‑tRNA synthetase that ligates glycine to its cognate tRNA to ensure translational fidelity. Through its essential role in tRNA charging, GlyRS supports global protein synthesis, proteostasis, and cellular stress responses linked to translation demand. Perturbation of tRNA synthetase activity is associated with neuromuscular and peripheral nerve phenotypes, making GARS a useful node for studying mechanisms connecting translational control to neuronal maintenance. GARS expression and function are also relevant to mitochondrial–cytosolic coordination of protein homeostasis and cellular adaptation to metabolic or proteotoxic stress.

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

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

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