SIM2s CRISPR Activation Plasmid (h): sc-405609-ACT

Human SIM2 (single-minded family bHLH transcription factor 2) encodes isoforms including SIM2s, a basic helix–loop–helix PAS-domain transcriptional regulator that forms heterodimers with ARNT and binds CNS midline enhancer-like elements to modulate gene expression programs. SIM2s contributes to lineage specification and differentiation, influencing transcriptional networks linked to development, epithelial identity, and cellular homeostasis. Altered SIM2/SIM2s expression has been reported in multiple disease-relevant contexts, including solid tumors and neurodevelopmental phenotypes, where it may intersect with hypoxia-responsive, aryl hydrocarbon receptor–associated, and broader transcriptional control pathways. These properties make SIM2s a useful node for studying context-dependent transcriptional regulation and downstream pathway remodeling.

SIM2s CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SIM2 expression without altering the underlying DNA sequence.

SIM2s CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SIM2 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 SIM2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SIM2s expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SIM2 locus and enabling the study of SIM2s-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SIM2s pathway restoration in tumor cells with silenced or reduced SIM2 expression.

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