HEM1 CRISPR Activation Plasmid (h): sc-407541-ACT

NCKAP1L encodes HEM1, a hematopoietic-enriched component of the WAVE regulatory complex that links Rac GTPase signaling to Arp2/3-dependent actin polymerization. By coordinating branched actin assembly, HEM1 supports immune cell morphology changes required for migration, adhesion, phagocytosis, and immune synapse formation, integrating cues from chemokine and integrin pathways. Perturbation of NCKAP1L disrupts cytoskeletal remodeling and can alter leukocyte trafficking and activation, making it relevant to studies of immune dysregulation and inflammatory disease mechanisms. Because actin dynamics also influence receptor signaling and vesicular transport, HEM1 is frequently investigated in pathways governing cell motility, polarity, and endocytosis in hematopoietic contexts.

HEM1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous NCKAP1L expression without altering the underlying DNA sequence.

HEM1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the NCKAP1L 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 NCKAP1L transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous HEM1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native NCKAP1L locus and enabling the study of HEM1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of HEM1 pathway restoration in tumor cells with silenced or reduced NCKAP1L expression.

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