IFT20 CRISPR Activation Plasmid (h): sc-418576-ACT

IFT20 (intraflagellar transport 20) encodes a core component of the intraflagellar transport B machinery that supports primary cilium assembly and maintenance, coordinating the trafficking of membrane and signaling proteins required for ciliogenesis. In addition to its ciliary role, IFT20 localizes to the Golgi and participates in vesicular sorting and polarized transport, linking secretory pathway dynamics to cilium-dependent signaling outputs. Through these functions, IFT20 influences pathways commonly organized at the primary cilium, including Hedgehog and other receptor-mediated signaling cascades that regulate proliferation and differentiation. Dysregulated IFT20 activity has been associated with cilia-related cellular phenotypes relevant to ciliopathy biology and with broader defects in cell signaling and tissue homeostasis.

IFT20 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IFT20 expression without altering the underlying DNA sequence.

IFT20 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IFT20 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 IFT20 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IFT20 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IFT20 locus and enabling the study of IFT20-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IFT20 pathway restoration in tumor cells with silenced or reduced IFT20 expression.

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