ASS1 CRISPR Activation Plasmid (h): sc-401923-ACT

Human ASS1 (argininosuccinate synthase 1) encodes a cytosolic enzyme that catalyzes the ATP-dependent condensation of citrulline and aspartate to form argininosuccinate, a central step in the urea cycle and arginine biosynthesis. By regulating nitrogen disposal and arginine availability, ASS1 influences cellular amino acid homeostasis, nitric oxide metabolism, and downstream polyamine synthesis. Altered ASS1 expression is linked to inherited urea cycle dysfunction and can reshape metabolic dependencies in tumor cells, where arginine auxotrophy and rewired one-carbon and nucleotide metabolism are frequently observed. These features make ASS1 a useful node for studying metabolic pathway regulation, nutrient stress responses, and genotype–phenotype relationships in human cell models.

ASS1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous ASS1 expression without altering the underlying DNA sequence.

ASS1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ASS1 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 ASS1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous ASS1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ASS1 locus and enabling the study of ASS1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of ASS1 pathway restoration in tumor cells with silenced or reduced ASS1 expression.

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