Rho T1 CRISPR Activation Plasmid (h): sc-401601-ACT

RHOT1 encodes Rho T1 (Miro1), an atypical Rho-family GTPase anchored to the outer mitochondrial membrane that coordinates mitochondrial trafficking, positioning, and quality control. Through Ca2+-binding EF-hand domains and interactions with kinesin/dynein adaptors, RHOT1 helps couple mitochondria to microtubule-based transport and regulates mitochondrial dynamics, bioenergetic homeostasis, and mitophagy, including PINK1/PRKN-associated pathways. Dysregulated RHOT1 function has been linked to altered mitochondrial distribution and impaired organelle turnover, processes implicated in neurodegeneration, neuropathies, and other disorders with mitochondrial stress phenotypes. As a mitochondrial transport regulator, RHOT1 is frequently studied in neuronal models, oxidative stress paradigms, and assays of mitochondrial network integrity and respiration.

Rho T1 CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous RHOT1 expression without altering the underlying DNA sequence.

Rho T1 CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the RHOT1 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 RHOT1 transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous Rho T1 expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native RHOT1 locus and enabling the study of Rho T1-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of Rho T1 pathway restoration in tumor cells with silenced or reduced RHOT1 expression.

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