IRAK-M CRISPR Activation Plasmid (h): sc-403219-ACT

IRAK3 encodes IRAK-M, a myeloid-enriched negative regulator of innate immune signaling that dampens Toll-like receptor and IL-1 receptor cascades by limiting IRAK1/IRAK4-dependent signal propagation from the MyD88 complex. By constraining downstream NF-κB and MAPK activation, IRAK-M helps shape cytokine and chemokine outputs and contributes to endotoxin tolerance and resolution of inflammatory responses. Altered IRAK3 expression has been linked to dysregulated inflammatory signaling in macrophages and monocytes and is frequently examined in contexts of chronic inflammation, immune suppression, and pathogen-driven immune evasion. These properties make IRAK-M a useful node for dissecting feedback control in innate immune pathways and transcriptional programs associated with myeloid cell state transitions.

IRAK-M CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous IRAK3 expression without altering the underlying DNA sequence.

IRAK-M CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the IRAK3 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 IRAK3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous IRAK-M expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native IRAK3 locus and enabling the study of IRAK-M-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of IRAK-M pathway restoration in tumor cells with silenced or reduced IRAK3 expression.

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