Bestrophin CRISPR Activation Plasmid (h): sc-404183-ACT

Human BEST1 encodes bestrophin-1, a Ca2+-activated anion channel predominantly localized to the basolateral membrane of retinal pigment epithelium. Bestrophin contributes to chloride and bicarbonate flux, epithelial ion and fluid homeostasis, and coupling of intracellular Ca2+ signals to membrane conductance that supports transepithelial transport processes. BEST1 activity intersects with pathways regulating membrane potential, cell volume, and retinal metabolism through regulation of ionic microenvironments. Genetic variation or dysregulation of BEST1 is associated with inherited retinal disorders, making it a useful target for studying RPE physiology and mechanisms of retinal degeneration in cellular models.

Bestrophin CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous BEST1 expression without altering the underlying DNA sequence.

Bestrophin CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the BEST1 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 BEST1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Bestrophin expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native BEST1 locus and enabling the study of Bestrophin-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Bestrophin pathway restoration in tumor cells with silenced or reduced BEST1 expression.

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