Crk-L CRISPR Activation Plasmid (h): sc-401097-ACT

CRKL encodes Crk-L, an SH2/SH3 adaptor protein that couples activated receptor and nonreceptor tyrosine kinases to downstream signaling complexes controlling cell adhesion, migration, proliferation, and survival. Crk-L participates in integrin- and growth factor–driven pathways, including focal adhesion dynamics and Ras/MAPK and PI3K signaling, by linking phosphorylated docking proteins to effector modules. It is a well-studied node in ABL-family kinase signaling and is frequently examined in contexts of altered tyrosine phosphorylation networks. Dysregulation of CRKL expression or signaling connectivity is associated with aberrant cell motility and oncogenic pathway activation, making it relevant for mechanistic studies of transformation and invasive phenotypes.

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

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

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