EYA4 CRISPR Activation Plasmid (h): sc-404355-ACT

EYA4 encodes a member of the Eyes Absent (EYA) family that functions as a transcriptional coactivator and phosphatase, integrating developmental transcription programs with signal-responsive regulation of cell fate. EYA4 participates in gene regulatory networks that influence differentiation, organogenesis, and sensory system maintenance, and it can couple to pathways controlling nuclear transcriptional output and stress-responsive signaling. In human biology, altered EYA4 activity or expression has been associated with phenotypes involving auditory function and cardiomyopathy, making it a relevant target for studying mechanisms of tissue homeostasis and dysfunction. Researchers commonly interrogate EYA4 to understand how transcriptional cofactors and phosphatase activities coordinate lineage-specific gene expression and cellular adaptation.

EYA4 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EYA4 expression without altering the underlying DNA sequence.

EYA4 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EYA4 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 EYA4 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EYA4 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EYA4 locus and enabling the study of EYA4-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EYA4 pathway restoration in tumor cells with silenced or reduced EYA4 expression.

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