LIN-28 CRISPR Activation Plasmid (h): sc-401164-ACT

Human LIN28A encodes LIN-28, an evolutionarily conserved RNA-binding protein that regulates post-transcriptional gene expression by modulating mRNA stability and translation and by selectively blocking maturation of let-7 family microRNAs. Through the LIN28/let-7 axis, LIN-28 influences developmental timing, maintenance of stem-like states, and metabolic reprogramming, with downstream effects on growth and differentiation programs. LIN28A activity intersects with miRNA biogenesis pathways and signaling networks that shape cell fate decisions, including contexts linked to proliferation and pluripotency. Dysregulated LIN28A/let-7 regulation has been associated with tumor biology and altered differentiation states, making it a useful target for mechanistic studies of oncogenic networks and developmental gene regulation.

LIN-28 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous LIN28A expression without altering the underlying DNA sequence.

LIN-28 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the LIN28A 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 LIN28A transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous LIN-28 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native LIN28A locus and enabling the study of LIN-28-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of LIN-28 pathway restoration in tumor cells with silenced or reduced LIN28A expression.

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