cSHMT CRISPR Activation Plasmid (h): sc-403678-ACT

Human SHMT1 encodes cytosolic serine hydroxymethyltransferase (cSHMT), a pyridoxal phosphate–dependent enzyme that catalyzes reversible serine-to-glycine conversion while generating 5,10-methylenetetrahydrofolate for one-carbon metabolism. cSHMT links folate-dependent nucleotide biosynthesis with methylation capacity by supplying one-carbon units to thymidylate and purine synthesis and coordinating redox-sensitive metabolic flux. Through its role in folate/one-carbon pathways, SHMT1 influences DNA replication and repair fidelity, epigenetic regulation, and cellular responses to nutrient availability. Dysregulation of this axis is frequently examined in models of proliferative stress and metabolic reprogramming, with implications for genomic stability–associated disease phenotypes.

cSHMT CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SHMT1 expression without altering the underlying DNA sequence.

cSHMT CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SHMT1 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 SHMT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous cSHMT expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SHMT1 locus and enabling the study of cSHMT-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of cSHMT pathway restoration in tumor cells with silenced or reduced SHMT1 expression.

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