Limd1 CRISPR Activation Plasmid (h): sc-404413-ACT

LIMD1 (LIM domains containing 1) encodes a scaffold protein that localizes to the nucleus and focal adhesion-associated complexes, integrating signaling cues that regulate transcription, cytoskeletal organization, and cell-cycle control. Limd1 participates in protein–protein interaction networks that influence Hippo pathway output and other growth-regulatory programs by modulating the availability and activity of transcriptional co-regulators. Through these roles, LIMD1 is commonly studied in contexts of altered proliferation, adhesion, and stress responses, where its dysregulation is frequently associated with tumor suppressor-like phenotypes. Functional interrogation of LIMD1 helps elucidate mechanisms linking cell contact, mechanotransduction, and gene expression control in human cells.

Limd1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous LIMD1 expression without altering the underlying DNA sequence.

Limd1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the LIMD1 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 LIMD1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Limd1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native LIMD1 locus and enabling the study of Limd1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Limd1 pathway restoration in tumor cells with silenced or reduced LIMD1 expression.

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