MAD1 CRISPR Activation Plasmid (h): sc-401885-ACT

Human MAD1L1 encodes mitotic arrest deficient 1 (MAD1), a core component of the spindle assembly checkpoint that safeguards chromosome segregation by monitoring kinetochore–microtubule attachment. MAD1 cooperates with MAD2 and other checkpoint factors to promote mitotic checkpoint complex formation and inhibit APC/C-CDC20 activity until proper biorientation is achieved, thereby limiting aneuploidy and chromosomal instability. Through its roles in mitotic timing and checkpoint signaling, MAD1L1 is frequently studied in pathways linking genome maintenance to cell cycle progression. Dysregulation of MAD1L1-associated checkpoint control has been implicated in chromosomal instability phenotypes observed across multiple cancer contexts and in models of impaired mitotic fidelity.

MAD1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous MAD1L1 expression without altering the underlying DNA sequence.

MAD1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the MAD1L1 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 MAD1L1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MAD1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native MAD1L1 locus and enabling the study of MAD1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MAD1 pathway restoration in tumor cells with silenced or reduced MAD1L1 expression.

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