RUFY4 CRISPR Activation Plasmid (h): sc-405000-ACT

RUFY4 (RUN and FYVE domain containing 4) encodes a cytosolic adaptor protein implicated in membrane trafficking events that couple small GTPase signaling to endosomal dynamics. Through its RUN and lipid-binding FYVE-related features, RUFY4 is associated with vesicular transport, endosome–lysosome organization, and spatial control of receptor and cargo routing that can influence innate immune signaling outputs. Expression patterns and pathway links suggest roles in myeloid and immune cell biology, including processes that shape inflammation and antigen handling. Dysregulated trafficking and immune signaling networks involving RUFY4 are relevant to studies of infection biology, inflammatory disorders, and tumor–immune interactions, where endosomal regulation can impact downstream pathway activation.

RUFY4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RUFY4 expression without altering the underlying DNA sequence.

RUFY4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RUFY4 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 RUFY4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RUFY4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RUFY4 locus and enabling the study of RUFY4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RUFY4 pathway restoration in tumor cells with silenced or reduced RUFY4 expression.

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