U2AF35 CRISPR Activation Plasmid (h): sc-404986-ACT

Human U2AF1 encodes U2AF35, a core component of the U2 auxiliary splicing factor that recognizes the 3′ splice site and helps recruit U2 snRNP during spliceosome assembly. By partnering with U2AF2 (U2AF65) and interacting with polypyrimidine tracts and splice junction features, U2AF35 contributes to alternative splicing decisions that shape mRNA isoform output, transcript stability, and protein diversity. U2AF1-dependent splicing programs intersect with RNA processing, transcriptional coupling, and cell-cycle control, making it a key node in post-transcriptional gene regulation. Recurrent U2AF1 alterations and dysregulated splicing signatures are associated with hematologic malignancy biology and broader RNA-splicing pathway perturbations, supporting its use in mechanistic studies of spliceosome function and disease-relevant splicing changes.

U2AF35 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous U2AF1 expression without altering the underlying DNA sequence.

U2AF35 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the U2AF1 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 U2AF1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous U2AF35 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native U2AF1 locus and enabling the study of U2AF35-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of U2AF35 pathway restoration in tumor cells with silenced or reduced U2AF1 expression.

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