Nebulin CRISPR Activation Plasmid (h): sc-404182-ACT

Human NEB encodes nebulin, a giant filamentous scaffold protein of skeletal muscle thin filaments that stabilizes F-actin, defines filament length, and supports sarcomere alignment and force transmission. Nebulin interfaces with troponin–tropomyosin regulatory complexes and Z-disc components, linking contractile activation to cytoskeletal organization during myofibrillogenesis and muscle maintenance. Altered NEB expression or function is associated with structural myopathies, including nemaline myopathy, and is relevant to studies of muscle weakness, fiber-type remodeling, and actin-dependent mechanobiology. These features make NEB a central target for probing sarcomere assembly, excitation–contraction coupling outputs, and actin cytoskeleton dynamics in human muscle models.

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

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

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