LGA CRISPR Activation Plasmid (h): sc-404294-ACT

Human GLS2 encodes the mitochondrial glutaminase isoform LGA, a key enzyme that converts glutamine to glutamate and ammonia to support carbon and nitrogen flux into the TCA cycle and linked biosynthetic pathways. Through regulation of glutamine utilization, GLS2 influences cellular redox balance via glutathione synthesis and impacts mitochondrial metabolism under nutrient stress. GLS2 activity intersects with amino acid sensing and stress-response programs, shaping proliferation and differentiation states across diverse cell types. Dysregulated GLS2/LGA expression has been associated with altered metabolic phenotypes observed in oncology and liver-related pathophysiology, making it a useful node for studying metabolic rewiring mechanisms.

LGA CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GLS2 expression without altering the underlying DNA sequence.

LGA CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GLS2 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 GLS2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous LGA expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GLS2 locus and enabling the study of LGA-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of LGA pathway restoration in tumor cells with silenced or reduced GLS2 expression.

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