N-Ras CRISPR Activation Plasmid (h): sc-400180-ACT

NRAS encodes the human small GTPase N-Ras, a membrane-associated molecular switch that cycles between GDP- and GTP-bound states to relay signals from receptor tyrosine kinases and other upstream inputs. Active N-Ras regulates core growth and survival pathways including RAF–MEK–ERK (MAPK) and PI3K–AKT, influencing proliferation, differentiation, and cytoskeletal dynamics. Dysregulated NRAS signaling is frequently studied in oncogenic contexts where altered pathway flux promotes aberrant transcriptional programs and cellular fitness. As a central node in Ras-driven signaling, NRAS is widely used to interrogate pathway rewiring, feedback regulation, and context-dependent responses to extracellular cues.

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

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

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