MLH3 CRISPR Activation Plasmid (m): sc-432245-ACT

Mlh3 encodes MLH3, a MutL family DNA mismatch repair factor that forms a heterodimer with MLH1 and contributes to genome maintenance through mismatch repair and meiotic recombination. In meiosis, MLH1–MLH3 promotes class I crossover formation, while in somatic cells it participates in post-replicative surveillance that limits mutation accumulation and preserves chromosomal stability. MLH3 also interfaces with pathways responding to replication stress and DNA damage, linking repair capacity to cell cycle progression. Altered MLH3 function has been associated with mismatch repair defects, genomic instability phenotypes, and susceptibility to mutation-driven disease processes relevant to cancer biology and reproductive genetics research.

MLH3 CRISPR Activation Plasmid (m) provides a targeted, non-destructive approach to upregulating endogenous Mlh3 expression without altering the underlying DNA sequence.

MLH3 CRISPR Activation Plasmid (m) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the Mlh3 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 Mlh3 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous MLH3 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native Mlh3 locus and enabling the study of MLH3-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of MLH3 pathway restoration in tumor cells with silenced or reduced Mlh3 expression.

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