RBM35A CRISPR Activation Plasmid (h2): sc-405645-ACT-2

Human ESRP1 (epithelial splicing regulatory protein 1; RBM35A) encodes an RNA-binding splicing factor that recognizes GU-rich motifs to control alternative splicing programs characteristic of epithelial cells. ESRP1 helps maintain epithelial identity by coordinating isoform selection in transcripts involved in cell–cell adhesion, cytoskeletal organization, polarity, and epithelial–mesenchymal transition (EMT)-linked signaling networks, including pathways intersecting with FGFR and CD44 isoform regulation. Dysregulated ESRP1-mediated splicing has been associated with altered EMT states and tumor progression phenotypes in multiple epithelial-derived cancers, implicating it as a key node in RNA processing changes observed during metastasis-related remodeling. Gene editing of ESRP1 enables mechanistic studies of splicing-dependent regulation, isoform-resolved transcriptomics, and functional interrogation of EMT and epithelial differentiation programs in human cell models.

RBM35A CRISPR Activation Plasmid (h2) provides a targeted, non-destructive approach to upregulating endogenous ESRP1 expression without altering the underlying DNA sequence.

RBM35A CRISPR Activation Plasmid (h2) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the ESRP1 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 ESRP1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous RBM35A expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native ESRP1 locus and enabling the study of RBM35A-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of RBM35A pathway restoration in tumor cells with silenced or reduced ESRP1 expression.

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