spectrin β II CRISPR/Cas9 KO Plasmid (h): sc-401818

Overview

SPTBN1 encodes spectrin βII, a core component of the spectrin–actin cytoskeletal network that supports plasma membrane stability, organizes specialized membrane domains, and links membrane proteins to cortical actin. Spectrin βII contributes to cell polarity, vesicle trafficking, and mechanotransduction, and participates in processes such as neurite outgrowth, axon initial segment organization, and maintenance of epithelial architecture. Through interactions with ankyrins and ion channels, it helps coordinate cytoskeletal scaffolding with signaling and transport pathways. Disruption or altered regulation of SPTBN1 has been associated with neurodevelopmental phenotypes and cytoskeleton-related cellular dysfunction, making it relevant for studies of neuronal connectivity and membrane-cortex organization.

spectrin β II CRISPR/Cas9 KO Plasmid (h) is a pool of plasmids designed for targeted disruption of the SPTBN1 gene in human cell lines. Each plasmid in the pool co-expresses a unique sgRNA, targeting a distinct site within the SPTBN1 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, spectrin β II 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 SPTBN1 target site.
When co-transfected with spectrin β II CRISPR/Cas9 KO Plasmid (h):

• The PuroR-RFP cassette integrates at the Cas9 cut site via HDR, disrupting the SPTBN1 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 SPTBN1 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 SPTBN1 exon(s) critical for spectrin β II 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.