Mer CRISPR Activation Plasmid (h): sc-402654-ACT

Human RYK (related to tyrosine kinase) encodes a catalytically impaired receptor tyrosine kinase–like protein that functions as a co-receptor in Wnt signaling, particularly within non-canonical Wnt/planar cell polarity pathways. Through ligand interactions with WNT5A and related cues, RYK modulates cytoskeletal dynamics, cell polarity, and migratory behavior, influencing tissue patterning and neuronal guidance programs. Altered RYK expression or signaling output has been associated with dysregulated developmental processes and with phenotypes relevant to oncogenic invasion and metastasis biology. These properties make RYK a useful node for dissecting context-specific Wnt pathway wiring and cell-state transitions in human cell models.

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

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

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