Rootletin CRISPR Activation Plasmid (h): sc-401646-ACT

CROCC encodes rootletin, a large coiled-coil cytoskeletal protein that polymerizes into ciliary rootlets and contributes to centrosome cohesion, basal body docking, and ciliogenesis. Rootletin scaffolds centrosomal architecture and supports microtubule organization required for cell-cycle progression and maintenance of epithelial polarity. Through its role in primary cilium formation and centrosome integrity, CROCC is relevant to pathways governing cilia-dependent signaling and mitotic fidelity. Dysregulation of these processes is associated with ciliopathy-related phenotypes and broader defects in tissue homeostasis linked to abnormal ciliary structure or centrosome function.

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

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

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