Nogo CRISPR/Cas9 KO Plasmid (h): sc-400819

Overview

RTN4 encodes Nogo (also known as neurite outgrowth inhibitor), a reticulon family protein enriched in the endoplasmic reticulum and, for some isoforms, present at the cell surface where it modulates neurite extension. Nogo signaling engages receptors such as RTN4R/NgR1 and co-receptors to activate RhoA–ROCK pathways that remodel the actin cytoskeleton, influencing axon guidance, synaptic plasticity, and neuronal network stability. Beyond the nervous system, RTN4 participates in ER membrane curvature, protein trafficking, and cellular stress responses, linking it to processes that affect cell morphology and survival. Dysregulated Nogo/RTN4 activity has been implicated in neurodegeneration, impaired CNS repair, and altered tumor cell migration, making it relevant for mechanistic studies of neuronal inhibition and cytoskeletal signaling.

Nogo CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the RTN4 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the RTN4 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, Nogo HDR Plasmid (h) 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 RTN4 target site.
When co-transfected with Nogo CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the RTN4 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 RTN4 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 RTN4 exon(s) critical for Nogo 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.