MTHFD1 CRISPR Activation Plasmid (h): sc-403353-ACT

Human MTHFD1 encodes a cytosolic, trifunctional folate-dependent enzyme that catalyzes interconversion of 10-formyl-, 5,10-methenyl-, and 5,10-methylene-tetrahydrofolate, supplying one-carbon units required for de novo purine and thymidylate biosynthesis and methylation reactions. By coupling folate metabolism to nucleotide production, MTHFD1 supports DNA replication, repair, and cell-cycle progression, especially under conditions of proliferative demand. Dysregulation of one-carbon metabolism has been linked to genomic instability and altered epigenetic states, making MTHFD1 a frequently studied node in metabolic rewiring and stress adaptation. Genetic variation or perturbed expression of MTHFD1 is also associated with folate-related phenotypes and susceptibility to disorders involving impaired nucleotide balance and methyl donor availability.

MTHFD1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MTHFD1 expression without altering the underlying DNA sequence.

MTHFD1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MTHFD1 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 MTHFD1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MTHFD1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MTHFD1 locus and enabling the study of MTHFD1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MTHFD1 pathway restoration in tumor cells with silenced or reduced MTHFD1 expression.

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