NELF-E CRISPR Activation Plasmid (h): sc-401932-ACT

Human NELFE encodes NELF-E, an RNA-binding subunit of the negative elongation factor (NELF) complex that enforces promoter-proximal pausing of RNA polymerase II and shapes early transcription elongation. Through coordination with DSIF and P-TEFb, NELF-E helps regulate transcriptional timing, stimulus-responsive gene programs, and co-transcriptional RNA processing, including coupling to 5′ end events. This control is central to pathways governing cell cycle progression, differentiation, and stress responses, where altered elongation dynamics can remodel gene expression networks. Dysregulation of transcriptional pausing and elongation control, including NELF complex components, has been linked to aberrant growth and lineage programs observed across diverse disease-relevant contexts, making NELFE a useful node for mechanistic studies of transcription regulation.

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

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

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