BBS8 CRISPR Activation Plasmid (h): sc-404755-ACT

TTC8 encodes BBS8, a core component of the BBSome complex that coordinates cargo trafficking to and from the primary cilium, supporting ciliary membrane protein sorting and intraflagellar transport–linked processes. Through regulation of cilia-dependent signaling, including Sonic hedgehog and GPCR trafficking, BBS8 helps maintain cellular polarity and sensory signaling across multiple tissues. Genetic disruption or dysfunction of BBS8 is associated with Bardet–Biedl syndrome and related ciliopathies, which present with pleiotropic phenotypes consistent with impaired ciliary structure and signaling. In human cell models, perturbing TTC8 expression provides a tractable approach to study ciliary proteostasis, vesicular transport, and pathway rewiring downstream of altered ciliary signaling.

BBS8 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous TTC8 expression without altering the underlying DNA sequence.

BBS8 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the TTC8 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 TTC8 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous BBS8 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native TTC8 locus and enabling the study of BBS8-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of BBS8 pathway restoration in tumor cells with silenced or reduced TTC8 expression.

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