Fatso CRISPR Activation Plasmid (m): sc-424024-ACT

Mouse Fto encodes the Fatso protein, an Fe(II)/2-oxoglutarate–dependent dioxygenase that functions as an RNA demethylase regulating reversible N6-methyladenosine (m6A) and related RNA methyl marks. By modulating RNA splicing, stability, and translation, Fatso influences gene expression programs linked to energy homeostasis, adipogenesis, and metabolic signaling pathways. Fto activity intersects with nutrient-sensing networks and impacts cell-state transitions through post-transcriptional control of transcripts involved in mitochondrial function and stress responses. Dysregulated Fto expression or activity has been associated with obesity-linked phenotypes, altered glucose metabolism, and broader cardiometabolic and neurobehavioral traits in model systems.

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

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

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