Tim44 CRISPR/Cas9 KO Plasmid (m): sc-423401

Overview

Timm44 encodes Tim44, a conserved component of the mitochondrial presequence translocase-associated motor at the inner membrane that helps couple protein import through the TIM23 complex to ATP-dependent pulling by mitochondrial Hsp70. By organizing the import machinery and supporting efficient matrix translocation, Tim44 contributes to mitochondrial proteostasis, oxidative metabolism, and maintenance of organelle function. Perturbation of TIM23 motor activity can trigger mitochondrial stress responses and secondary effects on apoptosis, reactive oxygen species handling, and energy-demanding cellular programs. Because mitochondrial import defects are implicated in diverse neuromuscular and neurodegenerative phenotypes, Timm44 is of interest for modeling how impaired biogenesis influences tissue vulnerability and metabolic disease mechanisms.

Tim44 CRISPR/Cas9 KO Plasmid (m) is a pool of plasmids designed for targeted disruption of the Timm44 gene in mouse cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the Timm44 locus, alongside the Streptococcus pyogenes Cas9 nuclease, and encodes GFP to enable fluorescent identification and enrichment of successfully transfected cells. This multi-guide strategy increases the likelihood of inducing frameshifts or deletions that produce a functional knockout, offering a more robust alternative to single-guide approaches. DSBs induced at multiple sites are resolved through non-homologous end joining (NHEJ) or, when used with the included HDR donor template, homology-directed repair (HDR) at a defined target site within the locus.

When used in conjunction with the RFP-expressing HDR donor, GFP and RFP fluorescence can be used together to distinguish transfected from edited cell populations, streamlining flow cytometry-based sorting and clone selection workflows.

Homology-Directed Repair (HDR) Donor — Puromycin Cassette with RFP Reporter

For applications requiring confirmed, selectable knockout clones, Tim44 HDR Plasmid (m) includes an HDR donor construct containing a puromycin resistance cassette (PuroR) and a red fluorescent protein (RFP) reporter, flanked by homology arms specific to a defined Timm44 target site.
When co-transfected with Tim44 CRISPR/Cas9 KO Plasmid (m):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the Timm44 open reading frame.
• RFP fluorescence provides an immediate visual indicator of successful integration, enabling fluorescence-based identification or sorting of edited cells prior to or alongside puromycin selection.
• Successfully edited cells are confirmed through puromycin resistance, substantially reducing clone screening burden.
• This selection strategy is ideal for generating stable, clonal KO cell lines for downstream functional studies, drug screening, or model development.

Cre-lox Cassette Removal System

The HDR donor construct features loxP sites flanking the PuroR-RFP selection cassette to allow clean marker removal following clone confirmation. Transient expression of Cre recombinase via the included Cre Vector: sc-418923 excises the cassette, leaving a minimal residual loxP site within the Timm44 locus and eliminating potential confounding effects on downstream assays.
This two-step approach:

• Minimizes disruption to local chromatin architecture and neighboring regulatory elements
• Restores a near-native genomic context at the edited locus
• Enables reuse of the puromycin selection strategy in the same cell line for additional edits

Key Features

• gRNA targeting Timm44 exon(s) critical for Tim44 function
• Co-expression of SpCas9 and sgRNA from a single plasmid for simplified delivery
• HDR donor with puromycin resistance for positive clone selection
• loxP-flanked PuroR cassette with Cre recombinase vector for seamless marker removal
• Supplied ready to use for delivery by transfection

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