SPA-L3 CRISPR Activation Plasmid (h): sc-411666-ACT

Human SIPA1L3 encodes SPA-L3, a multidomain signaling scaffold that interacts with small GTPase regulatory networks and organizes protein assemblies at the plasma membrane and cortical cytoskeleton. Through its association with Rap/Ras pathway components and actin-linked complexes, SPA-L3 is positioned to influence cell polarity, adhesion, and dynamic remodeling of the cytoskeleton that shape migration and morphogenesis. Altered regulation of these processes is frequently implicated in invasive behavior and aberrant tissue architecture, making SIPA1L3 a useful locus for studying pathway rewiring in disease-relevant cellular states. SIPA1L3 expression and network connectivity are therefore informative in models interrogating membrane-proximal signaling, cytoskeletal control, and phenotypic plasticity.

SPA-L3 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SIPA1L3 expression without altering the underlying DNA sequence.

SPA-L3 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SIPA1L3 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 SIPA1L3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous SPA-L3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SIPA1L3 locus and enabling the study of SPA-L3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of SPA-L3 pathway restoration in tumor cells with silenced or reduced SIPA1L3 expression.

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