NELF-A CRISPR Activation Plasmid (h): sc-401058-ACT

NELFA encodes NELF-A, a core subunit of the negative elongation factor (NELF) complex that cooperates with DSIF to establish promoter-proximal pausing of RNA polymerase II and regulate transcriptional elongation. Through this checkpoint, NELF-A helps coordinate rapid stimulus-responsive gene programs, coupling transcription initiation with productive elongation and RNA processing in the nucleus. NELF-mediated pausing influences chromatin state and transcriptional fidelity, shaping pathways involved in cell-cycle control, differentiation, and stress responses. Dysregulation of transcriptional elongation control has been associated with aberrant gene expression programs in cancer and neurodevelopmental phenotypes, supporting the use of NELFA perturbation to study disease-relevant transcriptional circuitry.

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

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

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