PU.1/Spi1 CRISPR Activation Plasmid (h): sc-400547-ACT

SPI1 encodes PU.1 (Spi1), an ETS-family transcription factor that functions as a lineage-determining regulator of hematopoietic differentiation, particularly in myeloid and B-cell compartments. PU.1 binds conserved ETS motifs to coordinate transcriptional programs controlling progenitor commitment, antigen presentation, immunoglobulin signaling components, and innate immune effector genes, integrating cues from cytokine and growth factor pathways. Altered SPI1 dosage or network wiring perturbs hematopoietic homeostasis and is implicated in leukemogenesis and immune dysregulation through downstream effects on differentiation, proliferation, and inflammatory gene expression. As a central node in hematopoietic gene regulatory circuits, PU.1 is widely used to model transcription factor–driven cell fate decisions and to map enhancer-promoter connectivity in immune cells.

PU.1/Spi1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous SPI1 expression without altering the underlying DNA sequence.

PU.1/Spi1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the SPI1 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 SPI1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous PU.1/Spi1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native SPI1 locus and enabling the study of PU.1/Spi1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of PU.1/Spi1 pathway restoration in tumor cells with silenced or reduced SPI1 expression.

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