β-NAP CRISPR Activation Plasmid (h): sc-411126-ACT

Human AP3B2 encodes β-NAP, the neuron-enriched beta subunit of the adaptor protein complex 3 (AP-3) that mediates cargo selection and trafficking from the trans-Golgi network and endosomal compartments to lysosomes and related organelles. β-NAP supports formation and targeting of vesicles critical for synaptic vesicle biogenesis and regulated secretion, linking AP3B2 to endolysosomal sorting, membrane protein localization, and neuronal homeostasis. Perturbation of AP-3–dependent trafficking can alter neurotransmitter release and organelle maturation, making AP3B2 a relevant node for studying neurodevelopmental and neurodegeneration-associated mechanisms. AP3B2 expression and function are therefore widely used as readouts in pathways involving vesicular transport, synaptic function, and endosomal–lysosomal dynamics.

β-NAP CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous AP3B2 expression without altering the underlying DNA sequence.

β-NAP CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the AP3B2 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 AP3B2 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous β-NAP expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native AP3B2 locus and enabling the study of β-NAP-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of β-NAP pathway restoration in tumor cells with silenced or reduced AP3B2 expression.

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