EN1/Engrailed 1 CRISPR Activation Plasmid (h): sc-405900-ACT

EN1 (Engrailed 1) encodes a homeobox transcription factor that regulates patterning and cell-fate specification during embryonic development, with prominent roles in midbrain–hindbrain organization and neuronal differentiation. By binding cis-regulatory elements, EN1 coordinates gene expression programs that shape morphogenesis, axon guidance, and lineage maintenance, interfacing with developmental signaling networks including Wnt, FGF, and Hedgehog-associated transcriptional outputs. In adult tissues and disease models, altered EN1 expression has been linked to dysregulated differentiation states and aberrant transcriptional circuitry. These features make EN1 a useful node for studying developmental gene regulatory networks and context-dependent transcriptional control in human cell systems.

EN1/Engrailed 1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous EN1 expression without altering the underlying DNA sequence.

EN1/Engrailed 1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the EN1 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 EN1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous EN1/Engrailed 1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native EN1 locus and enabling the study of EN1/Engrailed 1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of EN1/Engrailed 1 pathway restoration in tumor cells with silenced or reduced EN1 expression.

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